Human monoclonal antibody against a costimulatory signal transduction molecule ailim and pharmaceutical use thereof

ABSTRACT

Immunization of human antibody-producing transgenic mice, which have been created using genetic engineering techniques, with AILIM molecule as an antigen resulted in various human monoclonal antibodies capable of binding to AILIM and capable of controlling a variety of biological reactions (for example, cell proliferation, cytokine production, immune cytolysis, cell death, induction of ADCC, etc.) associated with AILIM-mediated costimulatory signal (secondary signal) transduction. Furthermore, it has been revealed that the human monoclonal antibody is effective to treat and prevent various diseases associated with AILIM-mediated costimulatory signal transduction, being capable of inhibiting the onset and/or advancement of the diseases.

This application claims priority from Japanese Application No.2000-147116, filed May 18, 2000, and Japanese Application No.2001-99508, filed Mar. 30, 2001.

FIELD OF THE INVENTION

The present invention relates to human antibodies which bind to AILIM(activation inducible lymphocyte immunomodulatory molecule, alsoreferred to as ICOS (inducible co-stimulator); human monoclonalantibodies which bind to AILIM or a portion thereof; DNA encoding saidhuman monoclonal antibody or a portion thereof, or a portion of saidDNA; cells (including genetic recombinant cells) producing said humanmonoclonal antibody or a portion thereof; human monoclonal antibody or aportion thereof produced by said genetic recombinant cells;pharmaceutical composition comprising said human monoclonal antibody ora portion thereof; pharmaceutical composition comprising antibody toAILIM for treating disorders related to the delayed allergy; method foridentifying, quantitating or assaying substances that bind to AILIM orAILIM ligand; and kit used for said method.

BACKGROUND OF THE INVENTION

A living body of mammals has immune response systems that excludespathogenic microorganisms (viruses, bacteria, parasites, etc.) orforeign bodies (both are called “antigen” in the following) that haveinvaded the living body. One of them is called natural immune responsesystem, another acquired immune response system. The former is anexclusion mechanism comprising phagocytosis by phagocytes(polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack bynatural killer (NK) cells, and non-specific recognition such asopsonization of antigen by complements. The latter, acquired immuneresponse system, is an exclusion mechanism by lymphocytes (mainly, Tcells and B cells) that acquired the specificity to the antigen (namely,activated lymphocytes). B cells that acquired antigen specificity attackthe antigen existing outside of the cells through production ofantibodies specific to the antigen. T cells that acquired antigenspecificity (namely, activated T cells) are classified into helper Tcells and cytotoxic T cells (cytotoxic lymphocyte, CTL). The helper Tcells regulate a differentiation of B cells and a production ofantibodies, and destroy the antigen cooperating with phagocytes. Thelatter, CTLs attack virus-infected cells and so on by themselves(Experimental Medicine: SUPPLEMENT, “Bio Science Term Library,Immunity”, Yodosha, pp. 14-17 (1995)).

This acquisition of antigen specificity by T cells (namely, activationof T cells) is initiated through recognition by T cells the antigenpresented by antigen-presenting cells (APC) such as macrophage, B cells,or dendritic cells. Antigen-presenting cells process the antigens soincorporated and present these processed antigens through binding themto major histocompatibility complex (MHC). T cells receive primarysignal for activation of the cells (or acquisition of specificity) byrecognizing the processed antigens presented by antigen-presenting cellsthrough a complex between T cell receptor (TcR) and CD3 antigen existingon the surface of the cell membrane (TcR/CD3 complex).

However, the TcR/CD3 complex-mediated primary signal alone cannotactivate T cells sufficiently and leads to unresponsiveness or clonalanergy, so that the cells can not react with any stimulation receivedthereafter. The autocrine of interleukin 2 (IL-2) is necessary for Tcells to be activated, to be differentiated into antigen specific T cellclones, and to be proliferated. In clonal anergy, T cells areinactivated due to no production of IL-2 and such and no cell division.Namely, the activation of T cells accompanied by production of cytokinessuch as IL-2 requires the secondary signal following the first signalthrough TcR/CD3 complex. This secondary signal is called costimulatorysignal.

T cells receive this secondary signal and transmit it into the cells byinteracting (cell adhesion) with molecules other than MHC onantigen-presenting cells through molecules other than TcR/CD3 complex onthe T cell surface. This secondary signal avoids cell anergy (clonalanergy) and activates the cells.

Although some part of the mechanism of the secondary signal transmissionbetween antigen-presenting cells and lymphocytes such as T cells havenot yet been elucidated in detail, studies so far have revealed that animportant factor for the secondary signal transmission is theinteraction of CD28 (also named Tp44, T44, or 9.3 antigen), which is acell surface molecule expressed mainly on T cells and thymus cells, withCD80 (also named B7-1, B7, BB1, or B7/BB1), which is a cell surfacemolecule expressed on antigen-presenting cells (macrophages, monocytes,dendritic cells, etc.) and with CD86 (also named B7-2 or B70), which isalso a cell surface molecule on antigen-presenting cells (namely, celladhesion through the binding between these molecules). Moreover, it hasbeen experimentally elucidated that the interaction of Cytolytic Tlymphocyte associated antigen 4 (CTLA-4), whose expression is thought tobe enhanced depending on the secondary signal, with the CD80 (B7-1) andCD86 (B7-2) (namely, cell adhesion through the binding between thesemolecules) also plays an important role in the regulation of T cellactivation by the secondary signal. In other words, the regulation of Tcell activation by the transmission of the secondary signal involves atleast the interaction between CD28 and CD80/CD86, the enhancement ofCTLA-4 expression, which is thought to depend on the interaction, andthe interaction between CTLA-4 and CD80/CD86.

CD28 is known to be a costimulator molecule transmitting the secondarysignal (costimulatory signal) required for the activation of T cells andfor the avoidance of anergy. The secondary signal transmitted by bindingthis molecule to costimulator molecules, CD80 (B7-1) and CD86 (B7-2), onantigen-presenting cells (cell adhesion through the binding betweenthese molecules), stabilizes mRNA of Th1-type cytokines and consequentlypromotes production by T cells of a large amount of Th1-type cytokinessuch as IL-2, IFNγ, and TNFα. The expression of CTLA-4 is induced by theprimary signal transmitted through TcR/CD3, and the expression is alsoenhanced by the secondary signal transmitted by the binding between CD28and CD80. It is being revealed that CTLA-4 receives these signals towork to inhibit T cell function, which is contrary to the activation ofT cells by the secondary signal transmitted by CD28.

Human CD28 and CTLA-4 are type I glycoproteins whose molecular weightsare 44 kD and 41 to 43 kD, respectively. Both have animmunoglobulin-like domain, belong to the immunoglobulin superfamily,and have both function as a cell adhesion molecule and function as asignal transmission molecule.

Human CD28 forms a homodimer with a disulfide bond while CTLA-4 existsas a monomer. Both CD28 and CTLA-4 genes are located at “2q33” on humanchromosome and “1C” on mouse chromosome, and are composed of four (4)exons. Human CD28 and CTLA-4 are composed of 220 and 223 amino acids,respectively, including the leader sequences, and amino acid homologybetween them is 20 to 30%.

The ligands for CD28 and CTLA-4 are CD80 (B7-1) and CD86 (B7-2) in humanand mice. CTLA-4 has about 20 times as high affinity to both ligands asCD28. It has been elucidated that the amino acid sequence structures“MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)” conserved through animal species isimportant for the binding of CD28 and CTLA-4 to CD80 (B7-1). It has alsobeen reported that, when CD28 is stimulated, P13 kinase(phosphoinositide 3 kinase, P13K) associates with the phosphorylatedtyrosine residue in a partial sequence “YMNM (Tyr-Met-Asn-Met)” of CD28and that CD28 plays an important role in intracellular signaltransmission through this “YxxM” structure. Furthermore, it has beenreported that CTLA-4 also has a sequence represented by “YxxM,” namely“YVKM (Tyr-Val-Lys-Met)” in its cytoplasmic region and that, after beingstimulated, SYP associates with this sequence.

CD28 is expressed specifically in thymocytes and peripheral blood Tcells, and CTLA-4 is expressed specifically in activated T cells (CellEngineering: SUPPLEMENT, “Handbook of Adhesion Molecule”, Shujunsha, pp.93-102 (1994); ibid. pp. 120-136; Experimental Medicine: SUPPLEMENT,“BIO SCIENCE Term Library, Immunity”, Yodosha, pp. 94-98 (1995);Experimental Medicine: SUPPLEMENT, “BIO SCIENCE Term Library,Intracellular Signal Transduction”, Yodosha, pp. 58-59 (1997); NihonRinsho, Vol. 55, No. 6, pp. 215-220 (1997)).

In the regulation of T cell function (the activation and the inhibitionof function of T cells), the importance of interactions among multiplemolecules such as costimulator molecules (CD28, CD80 (B7-1), CD86(B7-2), etc.) and CTLA-4, which cooperates with them, has thus beenrecognized, and this has been drawn attention to the relationshipbetween these molecules and diseases, and the treatment of diseases byregulating the function of these molecules.

As described above, although a living body activates its acquired immuneresponse system against antigens that are foreign bodies to the livingbody (self), it also has immunological tolerance so as to show no immuneresponse against its own component (autoantigen). If immunologicaltolerance breaks down by some reason, immune response to the autoantigenoccurs, autoantigen-reactive T cells are induced by the same mechanismas mentioned above to fall into abnormal state of immunity, and variousautoimmune diseases are caused.

In other words, since non-stimulated antigen presenting cells (APC) innormal tissues do not express costimulatory molecules when the immunesystem of a living body is normal, T cells are in the unresponsivenessstate to maintain immunological tolerance even if autoantigen-reactive Tcells, which reacts with autoantigen, exist. It has been suggested thatin abnormal state of immunity, more autoantigen-reactive T cells areactivated due to abnormal excess and continuous expression ofcostimulatory molecules to thereby cause autoimmune diseases.

From such viewpoints recently, many attempts to treat various autoimmunediseases by modulating the transmission of costimulatory signals, forexample, the above-mentioned signal transmission between CD28/CTLA-4 andCD80/CD86, are proposed.

The results of such attempts have not yet clarified in detail themechanism of the T cell activation by interaction between costimulatorymolecules and the related molecules. Other unknown molecules may beinvolved in this mechanism.

Recently, there has been identified a novel co-stimulatory molecule likethe above-described “CD28” and “CTLA-4”, which is thought to carry outthe transduction of a second signal (co-stimulatory signal) essentialfor the activation of lymphocytes such as T cells, and functionalregulation coupled with said signal of activated lymphocytes such asactivated T cells. This molecule has been designated as AILIM(activation inducible lymphocyte immunomodulatory molecule) (in humans,mice and rats: Int. Immunol., Vol. 12, No. 1, p. 51-55, 2000), alsoreferred to as ICOS (inducible co-stimulator) (in humans: Nature, Vol.397, No. 6716, p. 263-266, 1999)).

On the other hand, novel molecules celled B7h, B7RP-1, GL50 or LICOSwhich are ligands (AILIM ligands) interacting with this costimulatorytransmission molecule AILIM (ICOS) have been identified very recently(Nature. Vol. 402, No. 6763, pp. 827-832, 1999; Nature Medicine, Vol. 5,No. 12, pp. 1365-1369, 1999; J. Immunology, Vol. 164, pp. 1653-1657,2000; Curr. Biol., Vol. 10, No. 6, pp. 333-336, 2000).

The identification of these two kinds of novel molecules, namely AILIM(ICOS) and B7RP-1 (B7h, GL50, LICOS), as the signal transduction pathwayfor the costimulatory signal essential for the above activation oflymphocytes such as T cells, and the control of the function ofactivated T cells, revealed that there is the novel third pathway by theinteraction between AILIM (ICOS) and B7RP-1 (B7h, GL50, LICOS), besidesthe known first and second signal pathways which are already knowntransduction pathway between CD28 and CD80 (B7-1)/CD86 (B7-2), and thatbetween CTLA4 and CD80 (B7-1)/CD 86 (B7-2).

Studies on the biological functions of these novel molecules, thefunction control of lymphocytes, such as T cells, through this thirdcostimulatory signal transduction by the molecules, and the relationshipbetween the novel signal transduction and diseases are in progress (J.Immunol., 166(1), pp. 1, 2001; J. Immunol., 165(9), pp. 5035, 2000;Biochem. Biophys. Res. Commun., 276(1), pp. 335, 2000; Immunity, 13(1),pp. 95, 2000; J. Exp. Med., 192(1), pp. 53, 2000; Eur. J. Immunol.,30(4), pp. 1040, 2000; WO 01/15732).

SUMMARY OF THE INVENTION

Specifically, an objective of the present invention is to revealbiological functions of the novel molecule AILIM, considered, like“CD28” and “CTLA-4”, as a molecule which transmits the secondary signal(costimulatory signal) essential for the activation of lymphocytes, suchas T cells, and which controls the functions of activated lymphocytes,such as activated T cells, by working with the signal; to revealrelationships between the expression of AILIM and diseases; and toprovide a method and a pharmaceutical which inhibit the development ofthe various diseases dependent on the expression pattern of AILIM orwhich treat the diseases by controlling the biological functions of theAILIM using the medical and pharmaceutical methods (for example, a drugsuch as a monoclonal antibody and a low molecular compound).

To achieve the above-described purposes, the present inventors haveactively pursued studies on human antibodies (particularly humanmonoclonal antibodies) against mammalian AILIMs (particularly humanAILIM), and as a result, by immunizing transgenic mice prepared usinggenetic recombination techniques so as to produce human antibodies withAILIM (specifically cell membrane fraction of cells expressing humanAILIM), succeeded first in the world in preparing a variety ofmonoclonal antibodies which bind to human AILIM, particularly thosewhich bind to human AILIM that regulate signal transduction mediated byhuman AILIM.

Since antibodies (particularly monoclonal antibodies) of this inventionare derived from humans, they do not induce any severe immune rejectiondue to the immunogenicity against humans, HAMA (human anti-mouseantigenicity), in the host at all, which has been a big problem (sideeffect) in therapy using antibody pharmaceuticals comprising antibodiesderived from non-human mammals such as mice, and thus dramaticallyenhancing the value of antibody as medicine.

Therefore, human antibodies (particularly human monoclonal antibodies)which bind to mammalian AILIMs (particularly human AILIM) of thisinvention and pharmaceutical compositions comprising said humanantibodies (particularly human monoclonal antibodies) are useful asdrugs to control, with no induction of immune rejection due to HAMA inthe host, various physiological reactions related to the transduction ofco-stimulatory signal to AILIM-expressing cells mediated by AILIM (forexample, proliferation of AILIM-expressing cells, cytokine production byAILIM-expressing cells, immune cytolysis or apoptosis ofAILIM-expressing cells, and activity to induce antibody-dependentcytotoxicity to AILIM-expressing cells, and so on), and/or are alsouseful as drugs to suppress and prevent development of symptoms and/orprogress of various disorders related to the signal transductionmediated by said AILIM, and as medicine to treat or prevent saiddisorder.

Specifically, pharmaceutical compositions according to this inventionare able to control (suppress or stimulate) proliferation ofAILIM-expressing cells or production of cytokine (for example,interferon g or interleukin 4, etc.) by AILIM-expressing cells, therebyenabling suppression of various pathological conditions and treatment orprevention of various disorders caused by diverse physiologicalphenomena related to signal transduction mediated by AILIM.

Use of pharmaceutical compositions according to this invention enablessuppression, prevention and/or treatment of, for example, variousdisorders (for example, rheumatoid arthritis, multiple sclerosis,autoimmune thyroiditis, allergic contact-type dermatitis, chronicinflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin-dependent diabetes mellitus, psoriasis, etc.)classified into autoimmune or allergic disorders (particularlyautoimmune disease and delayed allergy caused by cellular immunity);arthropathia (for example, rheumatoid arthritis (RA) and osteoarthritis(OA)), inflammation (e.g., hepatitis); graft versus host reaction (GVHreaction); graft versus host disease (GVHD); immune rejectionaccompanying transplantation (homoplasty or heteroplasty) of a tissue(tissues such as skin, cornea, bone, etc.) or organ (liver, heart, lung,kidney, pancreas, etc.); immune response triggered by a foreign antigenor autoantigen (for example, production of antibodies against saidantigen, cell proliferation, production of cytokines); and disorderspossibly caused by the abnormal intestinal immunity (specificallyinflammatory intestinal disorders (particularly clone disease andulcerative colitis) and alimentary allergy).

Furthermore, in the field of suppression/treatment of immune rejectionaccompanying transplantation of above-described tissues and organs, itis possible to augment the suppressive effect on transplant rejection ofknown immunosuppressant by using the pharmaceutical composition of thisinvention together with said drugs which have been utilized forsuppression of immune rejection in such a transplantation treatment.

Moreover, the pharmaceutical composition of the present invention can beapplied for treating or preventing, any inflammatory diseases to whichvarious steroids are indicated as antiphlogistic.

The pharmaceutical composition of the present invention can be appliedto inflammatory disease for example, inflammation accompanying variousarthritis (for example, rheumatoid arthritis, osteoarthritis),pneumonia, hepatitis (including viral hepatitis), inflammationaccompanying infectious diseases, inflammatory bowel diseases,intestinal enteritis, nephritis (inflammation accompanying glomerularnephritis, nephrofibrosis), gastritis, angiitis, pancreatitis,peritonitis, bronchitis, myocarditis, cerebritis, inflammation inpostischemic reperfusion injury (myocardial ischemic reperfusioninjury), inflammation attributed to immune rejection aftertransplantation of tissue and organ, burn, various skin inflammation(psoriasis, allergic contact-type dermatitis, lichen planus which ischronic inflammatory skin disease), inflammation in multiple organfailure, inflammation after operation of PTCA or PTCR, and inflammationaccompanying arteriosclerosis, and autoimmune thyroiditis.

In addition, by using a method for identifying substances that bind toAILIM or AILIM ligand, which is one of the present inventions, itbecomes possible to screen to select pharmaceuticals (chemical syntheticcompounds or antibodies) with potential activity to treat variousdisorders by binding to AILIM or AILIM ligands to regulate signaltransduction mediated by interaction of them.

Specifically, the present invention is the invention described from thefollowing (1) to (108).

(1) A human antibody which binds to AILIM.

(2) The human antibody of (1), wherein said AILIM is derived from human.

(3) A human monoclonal antibody which binds to AILIM or a portionthereof.

(4) The human monoclonal antibody or a portion thereof of (3), whereinsaid AILIM is derived from human.

(5) The human monoclonal antibody or a portion thereof of (3) or (4),wherein said human monoclonal antibody has an activity to inhibit asignal transduction into a cell mediated by AILIM.

(6) The human monoclonal antibody or a portion thereof of (5), whereinsaid activity to inhibit a signal transduction is (a) or (b) of thefollowings:

(a) activity to inhibit proliferation of AILIM-expressing cells, or

(b) activity to inhibit cytokine production from AILIM-expressing cells.

(7) The human monoclonal antibody or a portion thereof of (6), whereinsaid cytokine is one of the cytokines produced by Th1-type or Th2-type Tcell.

(8) The human monoclonal antibody or a portion thereof of (7), whereinsaid cytokine is interferon γ or interleukin 4.

(9) The human monoclonal antibody or a portion thereof of (5), whereinsaid human monoclonal antibody has an activity to prevent mixedlymphocyte reaction.

(10) The human monoclonal antibody or a portion thereof of (3) or (4),wherein said human monoclonal antibody has an activity to induce signaltransduction into a cell mediated by AILIM.

(1 1) The human monoclonal antibody or a portion thereof of (10),wherein said activity to induce signal transduction is (a) or (b) of thefollowings:

(a) activity to induce proliferation of AILIM-expressing cells, or

(b) activity to induce cytokine production from AILIM-expressing cells.

(12) The human monoclonal antibody or a portion thereof of (11), whereinsaid cytokine is one of the cytokines produced by Th1-type or Th2-type Tcell.

(13) The human monoclonal antibody or a portion thereof of (12), whereinsaid cytokine is interferon γ or interleukin 4.

(14) The human monoclonal antibody or a portion thereof of (3) or (4),wherein said human monoclonal antibody has an activity to induceantibody-dependent cytotoxicity to AILIM-expressing cells, and/or immunecytolysis or apoptosis of AILIM-expressing cells.

(15) The human monoclonal antibody or a portion thereof of (3) or (4),wherein the binding rate constant (ka) between said monoclonal antibodyand AILIM is 1.0×10³ (1/M.Sec) or more.

(16) The human monoclonal antibody or a portion thereof of (15), whereinsaid binding rate constant (ka) is 1.0×10⁴ (1/M.Sec) or more.

(17) The human monoclonal antibody or a portion thereof of (16), whereinsaid binding rate constant (ka) is 1.0×10⁵ (1/M.Sec) or more.

(18) The human monoclonal antibody or a portion thereof of (3) or (4),wherein the dissociation rate constant (kd) between said monoclonalantibody and AILIM is 1.0×10⁻³ (1/Sec) or less.

(19) The human monoclonal antibody or a portion thereof of (18), whereinsaid dissociation rate constant (kd) is 1.0×10⁻⁴ (1/Sec) or less.

(20) The human monoclonal antibody or a portion thereof of (19), whereinsaid dissociation rate constant (kd) is 1.0×10⁻⁵ (1/Sec) or less.

(21) The human monoclonal antibody or a portion thereof of (3) or (4),wherein the dissociation constant (Kd) between said monoclonal antibodyand AILIM is 1.0×10⁻⁶ (M) or less.

(22) The human monoclonal antibody or a portion thereof of (21), whereinsaid dissociation constant (Kd) is 1.0×10⁻⁷ (M) or less.

(23) The human monoclonal antibody or a portion thereof of (22), whereinsaid dissociation constant (Kd) is 1.0×10⁻⁸ (M) or less.

(24) The human monoclonal antibody or a portion thereof of (23), whereinsaid dissociation constant (Kd) is 1.0×10⁻⁹ (M) or less.

(25) The human monoclonal antibody or a portion thereof of (4), whereina V region DNA encoding a heavy chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinheavy chain V gene segment 1-02 or 3-13.

(26) The human monoclonal antibody or a portion thereof of (4), whereina V region DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinlight chain V gene segment L5 or A27.

(27) The human monoclonal antibody or a portion thereof of (25) or (26),wherein a V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from either the humanimmunoglobulin heavy chain V gene segment 1-02 or 3-13, and wherein a Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinlight chain V gene segment L5 or A27.

(28) The human monoclonal antibody or a portion thereof of (27), whereinthe V region DNA encoding a heavy chain variable region of said humanmonoclonal antibody is derived from the human immunoglobulin heavy chainV gene segment 1-02, and the V region DNA encoding a light chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin light chain V gene segment L5.

(29) The human monoclonal antibody or a portion thereof of (27), whereinthe V region DNA encoding a heavy chain variable region of said humanmonoclonal antibody is derived from the human immunoglobulin heavy chainV gene segment 3-13, and the V region DNA encoding a light chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin light chain V gene segment A27.

(30) The human monoclonal antibody or a portion thereof of (4), whereina heavy chain variable region of said human monoclonal antibody has anamino acid sequence defined in any of the following (a) through (f):

(a) amino acid sequence comprising amino acids from position 20 through117 of SEQ ID NO:28,

(b) amino acid sequence comprising amino acids from position 20 through117 of SEQ ID NO:28 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(c) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:32,

(d) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:32 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(e) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:36, or

(f) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:36, in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

(31) The human monoclonal antibody or a portion thereof of (4), whereina heavy chain polypeptide of said human monoclonal antibody has an aminoacid sequence defined in any of the following (a) through (f):

(a) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:28,

(b) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:28 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(c) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:32,

(d) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:32 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(e) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:36, or

(f) amino acid sequence comprising amino acids from position 20 through470 of SEQ ID NO:36 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(32) The human monoclonal antibody or a portion thereof of (4), whereina light chain variable region of said human monoclonal antibody has anamino acid sequence defined in any of the following (a) through (f):

(a) amino acid sequence comprising amino acids from position 23 through116 of SEQ ID NO:30,

(b) amino acid sequence comprising amino acids from position 23 through116 of SEQ ID NO:30 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(c) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:34,

(d) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:34 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(e) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:38, or

(f) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:38 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(33) The human monoclonal antibody or a portion thereof of (4), whereina light chain polypeptide of said human monoclonal antibody has an aminoacid sequence defined in any of the following (a) through (f):

(a) amino acid sequence comprising amino acids from position 23 through236 of SEQ ID NO:30,

(b) amino acid sequence comprising amino acids from position 23 through236 of SEQ ID NO:30 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(c) amino acid sequence comprising amino acids from position 21 through236 of SEQ ID NO:34,

(d) amino acid sequence comprising amino acids from position 21 through236 of SEQ ID NO:34 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(e) amino acid sequence comprising amino acids from position 21 through236 of SEQ ID NO:38, or

(f) amino acid sequence comprising amino acids from position 21 through236 of SEQ ID NO:38 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.

(34) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 20 through 117 according to SEQID NO:28, and

(b) a light chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 23 through 116 according to SEQID NO:30.

(35) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain polypeptide has an amino acid sequence from amino acid20 through 470 according to SEQ ID NO:28, and

(b) a light chain polypeptide has an amino acid sequence from amino acid23 through 236 according to SEQ ID NO:30.

(36) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 20 through 116 according to SEQID NO:32, and

(b) a light chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 21 through 116 according to SEQID NO:34.

(37) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 20 through 470 according to SEQ IDNO:32, and

(b) a light chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 21 through 236 according to SEQ IDNO:34.

(38) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 20 through 116 according to SEQID NO:36, and

(b) a light chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 21 through 116 according to SEQID NO:38.

(39) The human monoclonal antibody or a portion thereof of (4), whereinsaid human monoclonal antibody has the following characteristics (a) and(b):

(a) a heavy chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 20 through 470 according to SEQ IDNO:36, and

(b) a light chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 21 through 236 according to SEQ IDNO:38.

(40) The human monoclonal antibody or a portion thereof of any one of(3) through (29), wherein said human monoclonal antibody is a monoclonalantibody derived from a transgenic non-human mammal capable of producinghuman antibodies.

(41) The human monoclonal antibody or a portion thereof of (40), whereinsaid human monoclonal antibody is obtained by immunizing transgenicnon-human mammal capable of producing human antibody withAILIM-expressing cells, membrane fractions derived from said cells,whole molecules constituting AILIM or a portion thereof, or genesencoding AILIM or a portion thereof.

(42) The human monoclonal antibody or a portion thereof of (40) or (41),wherein said transgenic non-human mammal is a transgenic mouse.

(43) A DNA or a portion thereof encoding a polypeptide selected from thegroup consisting of (a) through (f) below:

(a) a polypeptide comprising the amino acid sequence from amino acid 20through 117 according to SEQ ID NO:28,

(b) a polypeptide comprising the amino acid sequence from amino acid 23through 116 according to SEQ ID NO:30,

(c) a polypeptide comprising the amino acid sequence from amino acid 20through 116 according to SEQ ID NO:32,

(d) a polypeptide comprising the amino acid sequence from amino acid 21through 116 according to SEQ ID NO:34,

(e) a polypeptide comprising the amino acid sequence from amino acid 20through 116 according to SEQ ID NO:36, and

(f) a polypeptide comprising the amino acid sequence from amino acid 21through 116 according to SEQ ID NO:38.

(44) A DNA or a portion thereof encoding a polypeptide selected from thegroup consisting of (a) through (f) below:

(a) a polypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:28,

(b) a polypeptide comprising the amino acid sequence from amino acids 23through 236 according to SEQ ID NO:30,

(c) a polypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:32,

(d) a polypeptide comprising the amino acid sequence from amino acids 21through 236 according to SEQ ID NO:34,

(e) a polypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:36, and

(f) a polypeptide comprising the amino acid sequence from amino acids 21through 236 according to SEQ ID NO:38.

(45) A DNA or a portion thereof selected from the group consisting of(a) through (f) below:

(a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

(c) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31,

(d) a DNA comprising the nucleotide sequence from nucleotides 88 through375 according to SEQ ID NO:33,

(e) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

(f) a DNA comprising the nucleotide sequence from nucleotides 93 through380 according to SEQ ID NO:37.

(46) A DNA or a portion thereof selected from a group consisting of (a)through (f) below:

(a) a DNA comprising the nucleotide sequence from nucleotides 69 through1481 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 39 through749 according to SEQ ID NO:29,

(c) a DNA comprising the nucleotide sequence from nucleotides 94 through1506 defined in SEQ ID NO:31,

(d) a DNA comprising the nucleotide sequence from nucleotides 28 through738 according to SEQ ID NO:33,

(e) a DNA comprising the nucleotide sequence from nucleotides 96 through1508 according to SEQ ID NO:35, and

(f) a DNA comprising the nucleotide sequence from nucleotides 33 through743 according to SEQ ID NO:37.

(47) A vector comprising the DNA of any one of (43) through (46).

(48) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, or

(c) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35.

(49) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 69 through1481 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, or

(c) a DNA comprising the nucleotide sequence from nucleotides 96 through1508 according to SEQ ID NO:35.

(50) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

(b) a DNA comprising the nucleotide sequence from nucleotides 88 through375 according to SEQ ID NO:33, or

(c) a DNA comprising the nucleotide sequence from nucleotides 93 through380 according to SEQ ID NO:37.

(51) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 39 through749 according to SEQ ID NO:29,

(b) a DNA comprising the nucleotide sequence from nucleotides 28 through738 according to SEQ ID NO:33, or

(c) a DNA comprising the nucleotide sequence from nucleotides 33 through743 according to SEQ ID NO:37.

(52) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27, and

(b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29.

(53) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 69 through1481 according to SEQ ID NO:27, and

(b) a DNA comprising the nucleotide sequence from nucleotides 39 through749 according to SEQ ID NO:29.

(54) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

(b) a DNA comprising the nucleotide sequence from nucleotides 88 through375 according to SEQ ID NO:33.

(55) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, and

(b) a DNA comprising the nucleotide sequence from nucleotides 28 through738 according to SEQ ID NO:33.

(56) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

(b) a DNA comprising the nucleotide sequence from nucleotides 93 through380 according to SEQ ID NO:37.

(57) The vector of (47) comprising a DNA according to the following (a)and (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 96 through1508 according to SEQ ID NO:35, and

(b) a DNA comprising the nucleotide sequence from nucleotides 33 through743 according to SEQ ID NO:37.

(58) A cell producing a human monoclonal antibody of any one of (3)through (42).

(59) The cell of (58), wherein said cell is a fused cell obtained byfusing B cell, derived from a mammal capable of producing said humanmonoclonal antibody, and myeloma cell derived from a mammal.

(60) A genetic recombinant host transformed by transferring a DNAdescribed below in (a) or a vector comprising said DNA, a DNA describedbelow in (b) or a vector comprising said DNA, or both DNAs describedbelow in (a) and (b) or a vector comprising both of said DNAs:

(a) a DNA encoding a heavy chain polypeptide or a portion thereof of amonoclonal antibody which binds to human AILIM; or

(b) a DNA encoding a light chain polypeptide or a portion thereof of amonoclonal antibody which binds to human AILIM.

(61) The genetic recombinant host of (60), wherein said monoclonalantibody is a human monoclonal antibody.

(62) The genetic recombinant host of (60) or (61), wherein said host isa mammalian cell.

(63) The genetic recombinant host of (60) or (61), wherein said host isa mammalian fertilized egg.

(64) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain polypeptide is one of the heavy chainpolypeptides selected from the group consisting of the following (a)through (c):

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 117 according to SEQ ID NO:28,

(b) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 116 according to SEQ ID NO:32, and

(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 116 according to SEQ ID NO:36.

(65) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain polypeptide is one of the heavy chainpolypeptide selected from the group consisting of the following (a)through (c):

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:28,

(b) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:32, and

(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:36.

(66) The genetic recombinant host of any one of (60) through (63),wherein said light chain polypeptide is one of the light chainpolypeptide selected from the group consisting of the following (a)through (c):

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 23 through 116 according to SEQ ID NO:30,

(b) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:34, and

(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.

(67) The genetic recombinant host of any one of (60) through (63),wherein said light chain polypeptide is one of the light chainpolypeptide selected from the group consisting of the following (a)through (c):

(a) a light chain polypeptide comprising the amino acid sequence fromamino acids 23 through 236 according to SEQ ID NO:30,

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:34, and

(c) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:38.

(68) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 117 according to SEQ ID NO:28, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 23 through 116 according to SEQ ID NO:30.

(69) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:28, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 23 through 236 according to SEQ ID NO:30.

(70) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 116 according to SEQ ID NO:32, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:34.

(71) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:32, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:34.

(72) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 116 according to SEQ ID NO:36, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.

(73) The genetic recombinant host of any one of (60) through (63),wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively:

(a) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:36, and

(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:38.

(74) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is a DNA definedin any of following (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

(c) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35.

(75) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is a DNA definedin any of following (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 69 through1481 according to SEQ ID NO:27,

(b) a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, and

(c) a DNA comprising the nucleotide sequence from nucleotides 96 through1508 according to SEQ ID NO:35.

(76) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said light chain polypeptide is a DNA definedin any of following (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

(b) a DNA comprising the nucleotide sequence from nucleotides 88 through375 according to SEQ ID NO:33, and

(c) a DNA comprising the nucleotide sequence from nucleotides 93 through380 according to SEQ ID NO:37.

(77) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said light chain polypeptide is a DNA asdefined in any of following (a) through (c):

(a) a DNA comprising the nucleotide sequence from nucleotides 39 through749 according to SEQ ID NO:29,

(b) a DNA comprising the nucleotide sequence from nucleotides 28 through738 according to SEQ ID NO:33, and

(c) a DNA comprising the nucleotide sequence from nucleotides 33 through743 according to SEQ ID NO:37.

(78) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is a DNA describedbelow in (a), and the DNA encoding said light chain polypeptide is a DNAas described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27, and

(b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29.

(79) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 69 through1481 according to SEQ ID NO:27, and

(b) a DNA comprising the nucleotide sequence from nucleotides 39 through749 according to SEQ ID NO:29.

(80) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

(b) a DNA comprising the nucleotide sequence from nucleotides 88 through375 V SEQ ID NO:33.

(81) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, and

(b) a DNA comprising the nucleotide sequence from nucleotides 28 through738 according to SEQ ID NO:33.

(82) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

(b) a DNA comprising the nucleotide sequence from nucleotides 93 through380 according to SEQ ID NO:37.

(83) The genetic recombinant host of any one of (60) through (63),wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

(a) a DNA comprising the nucleotide sequence from nucleotides 96 through1508 according to SEQ ID NO:35, and

(b) a DNA comprising the nucleotide sequence from nucleotides 33 through743 according to SEQ ID NO:37.

(84) A human monoclonal antibody or a portion thereof produced by agenetic recombinant host (provided excluding the case where said host isa fertilized egg) of any one of (60) through (62), or of any one of (64)through (83).

(85) A pharmaceutical composition comprising the human antibody of (1)or (2), and a pharmaceutically acceptable carrier.

(86) A pharmaceutical composition comprising the human monoclonalantibody or a portion thereof of any one of (3) to (42), and apharmaceutically acceptable carrier.

(87) A pharmaceutical composition comprising a human monoclonal antibodyor a portion thereof of (84), and a pharmaceutically acceptable carrier.

(88) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to inhibit signaltransduction into the cell mediated by AILIM.

(89) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to prevent proliferationof AILIM-expressing cells.

(90) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to prevent production ofa cytokine from AILIM-expressing cells.

(91) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to induce signaltransduction into a cell mediated by AILIM.

(92) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to induce proliferationof AILIM-expressing cells.

(93) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to induce production ofa cytokine from AILIM-expressing cells.

(94) The pharmaceutical composition of any one of (85) through (87),wherein said pharmaceutical composition is used to induceantibody-dependent cytotoxicity against AILIM-expressing cells, and/orimmune cytolysis or apoptosis of AILIM-expressing cells.

(95) A pharmaceutical composition for preventing, treating, orprophylaxis of delayed type allergy, comprising a substance having anactivity in modulating signal transduction mediated by AILIM, and apharmaceutically acceptable carrier.

(96) The pharmaceutical composition of (95), wherein the substance is aprotein substance.

(97) The pharmaceutical composition of (96), wherein the proteinsubstance is selected from the group consisting of:

a) an antibody which binds to AILIM or a portion thereof;

b) a polypeptide comprising the whole or a portion of an extracellularregion of AILIM;

c) a fusion polypeptide comprising the whole or a portion of anextracellular region of AILIM, and the whole or a portion of a constantregion of immunoglobulin heavy chain; and

d) a polypeptide which binds to AILIM.

(98) The pharmaceutical composition of (97), wherein said antibody thatbinds to AILIM is the human antibody of (1) or (2).

(99) The pharmaceutical composition of (97), wherein said antibody thatbinds to AILIM is the human monoclonal antibody of any one of (3)through (42).

(100) The pharmaceutical composition of (97), wherein said antibodyagainst AILIM is the human monoclonal antibody of (84).

(101) The pharmaceutical composition of (95), wherein the substance is anon-protein substance.

(102) The pharmaceutical composition of (101), wherein the non-proteinsubstance is DNA, RNA, or a chemically synthesized compound.

(103) A method for identifying substances that bind to AILIM or AILIMligand comprising the following processes:

(a) preparing an insoluble carrier on which the entire extracellularregion of AILIM or a portion thereof is immobilized;

(b) preparing a polypeptide comprising the whole extracellular region ofAILIM ligand or a portion thereof labeled with a labeling material thatemit a detectable signal;

(c) reacting the insoluble carrier in process(a) with the polypeptide inprocess (b);

(d) reacting the insoluble carrier of process (a), the polypeptide ofprocess (b) and said substance to each other in any arbitrary orders;

(e) detecting the signal emitted from said labeling material containedin the complex produced in process (c), and the signal emitted from saidlabeling material contained in the complex produced in process (d),respectively; and

(f) comparing the magnitude of each of signals detected in process (e).

(104) A method for identifying substances that bind to AILIM or AILIMligand comprising the following processes:

(a) preparing an insoluble carrier on which the entire extracellularregion of AILIM ligand or a portion thereof is immobilized;

(b) preparing a polypeptide comprising the whole extracellular region ofAILIM or a portion thereof labeled with a labeling material that emit adetectable signal;

(c) reacting the insoluble carrier in process (a) with the polypeptidein process (b);

(d) reacting the insoluble carrier of process (a), the polypeptide ofprocess (b) and said substance to each other in any arbitrary orders;

(e) detecting the signal emitted from said labeling material containedin the complex produced in process (c), and the signal emitted from saidlabeling material contained in the complex produced in process (d),respectively; and

(f) comparing the magnitude of each of signals detected in process (e).

(105) The method of (103) or (104), wherein said polypeptide comprisingthe whole extracellular region of AILIM or a portion thereof is a fusionpolypeptide comprising a polypeptide, comprising the whole extracellularregion of AILIM or a portion thereof, and the whole constant region ofimmunoglobulin heavy chain or a portion thereof.

(106) The method of (103) or (104), wherein said polypeptide comprisingthe whole extracellular region of AILIM ligand or a portion thereof is afusion polypeptide comprising a polypeptide, comprising the wholeextracellular region of AILIM ligand or a portion thereof, and the wholeconstant region of immunoglobulin heavy chain or a portion thereof.

(107) The method of any one of (103) through (106), wherein said AILIMis a human AILIM.

(108) The method of any one of (103) through (107), wherein said AILIMligand is a human AILIM ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows respective reactivities of anti-human IgG antibody,anti-human Igκ antibody and anti-human IgFc antibody to the humananti-human AILIM monoclonal antibody, analyzed by cell ELISA using aflow cytometer.

Panels (a) to (1) show respective results of the assays indicated below.

Panel (a): result of assay in which biotin-labeled anti-human IgGantibody as a secondary antibody was added in the absence of primaryantibody into the microplate where wild-type HPB-ALL cells had beenplated.

Panel (b): result of assay in which biotin-labeled anti-human Igκantibody as a secondary antibody was added in the absence of primaryantibody into the microplate where wild-type HPB-ALL cells had beenplated.

Panel (c): result of assay in which biotin-labeled anti-human IgFcantibody as a secondary antibody was added in the absence of primaryantibody into the microplate where wild-type HPB-ALL cells had beenplated.

Panel (d): result of assay in which human anti-human AILIM monoclonalantibody JMab-136 was used as a primary antibody and biotin-labeledanti-human IgG antibody was used as a secondary antibody.

Panel (e): result of assay in which human anti-human AILIM monoclonalantibody JMab-136 was used as a primary antibody and biotin-labeledanti-human Igκ antibody was used as a secondary antibody.

Panel (f): result of assay in which human anti-human AILIM monoclonalantibody JMab-136 was used as a primary antibody and biotin-labeledanti-human IgFc antibody was used as a secondary antibody.

Panel (g): result of assay in which human anti-human AILIM monoclonalantibody JMab-138 was used as a primary antibody and biotin-labeledanti-human IgG antibody was used as a secondary antibody.

Panel (h): result of assay in which human anti-human AILIM monoclonalantibody JMab-138 was used as a primary antibody and biotin-labeledanti-human Igκ antibody was used as a secondary antibody.

Panel (i): result of assay in which human anti-human AILIM monoclonalantibody JMab-138 was used as a primary antibody and biotin-labeledanti-human IgFc antibody was used as a secondary antibody.

Panel (j): result of assay in which human anti-human AILIM monoclonalantibody JMab-139 was used as a primary antibody and biotin-labeledanti-human IgG antibody was used as a secondary antibody.

Panel (k): result of assay in which human anti-human AILIM monoclonalantibody JMab-139 was used as a primary antibody and biotin-labeledanti-human Igκ antibody was used as a secondary antibody.

Panel (l): result of assay in which human anti-human AILIM monoclonalantibody JMab-139 was used as a primary antibody and biotin-labeledanti-human IgFc antibody was used as a secondary antibody.

The curve with open symbols in each panel corresponds to the result ofassay in which human anti-KLH monoclonal antibody was used as thecontrol antibody.

FIG. 2 shows a calibration curve with respect to human IgG monoclonalantibody (standard material) assayed by sandwich ELISA using anti-humanIgG antibody.

The vertical axis indicates fluorescence intensity, and the horizontalaxis indicates the concentration of the standard material.

FIG. 3 shows binding activities of various mouse anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “human” indicates the result of a bindingassay to the human AILIM-overexpressing recombinant CHO cell.

FIG. 4 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to human AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “human” indicates the result of a bindingassay to the human AILIM-overexpressing recombinant CHO cell.

FIG. 5 shows binding activities of various human anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “human” indicates the result of a bindingassay to the human AILIM-overexpressing recombinant CHO cell.

FIG. 6 shows binding activities of various human anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “human” indicates the result of a bindingassay to the human AILIM-overexpressing recombinant CHO cell.

FIG. 7 shows binding activities of rat anti-human AILIM monoclonalantibodies to mouse AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “mouse” indicates the result of a bindingassay to the mouse AILIM-overexpressing recombinant CHO cell.

FIG. 8 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to mouse AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “mouse” indicates the result of a bindingassay to the mouse AILIM-overexpressing recombinant CHO cell.

FIG. 9 shows binding activities of various human anti-human AILIMmonoclonal antibodies to mouse AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “mouse” indicates the result of a bindingassay to the mouse AILIM-overexpressing recombinant CHO cell.

FIG. 10 shows binding activities of various human anti-human AILIMmonoclonal antibodies to mouse AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “mouse” indicates the result of bindingassay to the mouse AILIM-overexpressing recombinant CHO cell.

FIG. 11 shows binding activities of various mouse anti-rat AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “rat” indicates the result of a bindingassay to the rat AILIM-overexpressing recombinant CHO cell.

FIG. 12 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to rat AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “rat” indicates the result of a bindingassay to the rat AILIM-overexpressing recombinant CHO cell.

FIG. 13 shows binding activities of various human anti-human AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “rat” indicates the result of a bindingassay to the rat AILIM-overexpressing recombinant CHO cell.

FIG. 14 shows binding activities of various human anti-human AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

The term “CHO” in the figure indicates the result of a binding assay tothe wild-type CHO cell, and “rat” indicates the result of a bindingassay to the rat AILIM-overexpressing recombinant CHO cell.

FIG. 15 shows proliferation activity of T cells derived from a normalhealthy person “donor A” in the assay for the activity of transducingcostimulatory signal by various mouse anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with mouse anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

FIG. 16 shows proliferation activity of T cells derived from a normalhealthy person “donor A” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

FIG. 17 shows proliferation activity of T cells derived from a normalhealthy person “donor B” in the assay for the activity of transducingcostimulatory signal by various mouse anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with mouse anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “JHC1” indicated result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 18 shows proliferation activity of T cells derived from a normalhealthy person “donor B” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

FIG. 19 shows proliferation activity of T cells derived from a normalhealthy person “donor B” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

FIG. 20 shows proliferation activity of T cells derived from a normalhealthy person “donor C” in the assay for the activity of transducingcostimulatory signal by various mouse anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with mouse anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 21 shows proliferation activity of T cells derived from a normalhealthy person “donor C” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

FIG. 22 shows proliferation activity of T cells derived from a normalhealthy person “donor C” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

FIG. 23 shows proliferation activity of T cells derived from a normalhealthy person “donor C” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“137”: human anti-human AILIM monoclonal antibody JMab137.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

FIG. 24 shows proliferation activity of T cells derived from a normalhealthy person “donor C” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 25 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of transducingcostimulatory signal by various mouse anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with mouse anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 26 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

FIG. 27 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

FIG. 28 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“137”: human anti-human AILIM monoclonal antibody JMab137.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

FIG. 29 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 30 shows proliferation activity of T cells derived from a normalhealthy person “donor E” in the assay for the activity of transducingcostimulatory signal by various mouse anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with mouse anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 31 shows proliferation activity of T cells derived from a normalhealthy person “donor E” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

FIG. 32 shows proliferation activity of T cells derived from a normalhealthy person “donor E” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

FIG. 33 shows proliferation activity of T cells derived from a normalhealthy person “donor E” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“137”: human anti-human AILIM monoclonal antibody JMab137.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

FIG. 34 shows proliferation activity of T cells derived from a normalhealthy person “donor E” in the assay for the activity of transducingcostimulatory signal by various human anti-human AILIM monoclonalantibodies using a microplate coated with anti-human CD3 monoclonalantibody together with human anti-human AILIM monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 35 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of various mouseanti-human AILIM monoclonal antibodies to transduce costimulatorysignal, when a solution of mouse anti-human AILIM monoclonal antibody(in liquid phase) was added alone to a microplate coated with anti-humanCD3 monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of mouse anti-human AILIMmonoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 36 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of various humananti-human AILIM monoclonal antibodies to transduce costimulatory signalwhen a solution of human anti-human AILIM monoclonal antibody (in liquidphase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“124”: human anti-human AILIM monoclonal antibody JMab124.

“125”: human anti-human AILIM monoclonal antibody JMab125.

“126”: human anti-human AILIM monoclonal antibody JMab126.

FIG. 37 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of various humananti-human AILIM monoclonal antibodies to transduce costimulatory signalwhen a solution of human anti-human AILIM monoclonal antibody (in liquidphase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

FIG. 38 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of various humananti-human AILIM monoclonal antibodies to transduce costimulatory signalwhen a solution of human anti-human AILIM monoclonal antibody (in liquidphase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“137”: human anti-human AILIM monoclonal antibody JMab137.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

FIG. 39 shows proliferation activity of T cells derived from a normalhealthy person “donor D” in the assay for the activity of various humananti-human AILIM monoclonal antibodies to transduce costimulatory signalwhen a solution of human anti-human AILIM monoclonal antibody (in liquidphase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of human anti-human AILIMmonoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 40 shows the amount of IFN-y produced in the culture supernatant ofT cells derived from a normal healthy person “donor B,” which werecultured in a microplate coated with mouse anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-g, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 41 shows the amount of IFN-g produced in the culture supernatant ofT cells derived from a normal healthy person “donor B,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

FIG. 42 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor B,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

FIG. 43 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor C,” which werecultured in a microplate coated with mouse anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “JHC1” indicates result of assay in which anti-humanCETP monoclonal antibody was used as the negative control, instead ofthe mouse anti-human AILIM monoclonal antibody.

FIG. 44 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor C,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“124”: human anti-human AILIM monoclonal antibody JMab124.

“125”: human anti-human AILIM monoclonal antibody JMab125.

“126”: human anti-human AILIM monoclonal antibody JMab126.

FIG. 45 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor C,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

FIG. 46 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor C,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“137”: human anti-human AILIM monoclonal antibody JMab137.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

FIG. 47 shows the amount of IFN-γ produced in the culture supernatant ofT cells derived from a normal healthy person “donor C,” which werecultured in a microplate coated with human anti-human AILIM monoclonalantibody together with anti-human CD3 monoclonal antibody.

The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

Other notations are as follows:

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 48 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor A”, with PBMCof a normal healthy person “donor D” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 49 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor A”, with PBMCof a normal healthy person “donor D” by various human anti-human AILIMmonoclonal antibodies in the proliferation test of the T cells throughthe mixed lymphocyte reactions (MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 50 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor D”, with PBMCof a normal healthy person “donor B” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 51 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor D”, with PBMCof a normal healthy person “donor B” by various human anti-human AILIMmonoclonal antibodies in the proliferation test of the T cells throughthe mixed lymphocyte reactions (MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 52 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor C”, with PBMCof a normal healthy person “donor A” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 53 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor C”, with PBMCof a normal healthy person “donor A” by various human anti-human AILIMmonoclonal antibodies in the proliferation test of the T cells throughthe mixed lymphocyte reactions (MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127,

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 54 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor E”, with PBMCof a normal healthy person “donor G” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86    antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

FIG. 55 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor E”, with PBMCof a normal healthy person “donor G” by various human anti-human AILIMmonoclonal antibodies in the proliferation test of the T cells throughthe mixed lymphocyte reactions (MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 56 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor F”, with PBMCof a normal healthy person “donor E” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86    antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

FIG. 57 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor F”, with PBMCof a normal healthy person “donor E” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 58 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor G”, with PBMCof a normal healthy person “donor F” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

-   “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86    antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody-   “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

FIG. 59 shows the inhibitory effect on T cell proliferation in the caseof culturing T cells from a normal healthy person “donor G”, with PBMCof a normal healthy person “donor F” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

The vertical axis indicates the amount of incorporation of [³H]thymidineas an index showing a level of cell proliferation, and the horizontalaxis shows the concentration of the test samples.

Each description in the figures shows the following.

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 60 shows the inhibitory effect of various control test substanceson the proliferation of T cells in the assay using mixed lymphocytereaction (MLR). T cells from a normal healthy person “donor A” wereco-cultured with PBMCs from a normal healthy person “donor D”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 61 shows the inhibitory effect of various human anti-human AILIMmonoclonal antibodies on the proliferation of T cells in the assay usingmixed lymphocyte reaction (MLR). T cells from a normal healthy person“donor A” were co-cultured with PBMCs from a normal healthy person“donor D” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 62 shows the inhibitory effect of various control test substanceson the proliferation of T cells in the assay using mixed lymphocytereaction (MLR). T cells from a normal healthy person “donor D” wereco-cultured with PBMCs from a normal healthy person “donor B”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 63 shows the inhibitory effect of various human anti-human AILIMmonoclonal antibodies on the proliferation of T cells in the assay usingmixed lymphocyte reaction (MLR). T cells from a normal healthy person“donor D” were co-cultured with PBMCs from a normal healthy person“donor B” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 64 shows the inhibitory effect of various control test substanceson the proliferation of T cells in the assay using mixed lymphocytereaction (MLR). T cells from a normal healthy person “donor C” wereco-cultured with PBMCs from a normal healthy person “donor A”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Each description in the figures shows the following.

-   “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86 antibody-   “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody FIG. 65 shows the    inhibitory effect of various human anti-human AILIM monoclonal    antibodies on the proliferation of T cells in the assay using mixed    lymphocyte reaction (MLR). T cells from a normal healthy person    “donor C” were co-cultured with PBMCs from a normal healthy person    “donor A” pre-cultured in the presence of human CTLA4-Ig chimeric    molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

“126”: human anti-human AILIM monoclonal antibody JMab126.

“127”: human anti-human AILIM monoclonal antibody JMab127.

“128”: human anti-human AILIM monoclonal antibody JMab128.

“135”: human anti-human AILIM monoclonal antibody JMab135.

“136”: human anti-human AILIM monoclonal antibody JMab136.

“137”: human anti-human AILIM monoclonal antibody JMab137.

FIG. 66 shows the inhibitory effect of various control test substanceson the proliferation of T cells in the assay using mixed lymphocytereaction (MLR). T cells from a normal healthy person “donor E” wereco-cultured with PBMCs from a normal healthy person “donor G”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Each description in the figures shows the following.

-   “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86    antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 67 shows the inhibitory effect of various human anti-human AILIMmonoclonal antibodies on the proliferation of T cells in the assay usingmixed lymphocyte reaction (MLR). T cells from a normal healthy person“donor E” were co-cultured with PBMCs from a normal healthy person“donor G” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

“138”: human anti-human AILIM monoclonal antibody JMab138.

“139”: human anti-human AILIM monoclonal antibody JMab139.

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab141.

FIG. 68 shows the inhibitory effect of various control test substanceson the proliferation of T cells in the assay using mixed lymphocytereaction (MLR). T cells from a normal healthy person “donor G” wereco-cultured with PBMCs from a normal healthy person “donor F”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Each description in the figures shows the following.

-   “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody-   “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86    antibody-   “SA12”: Anti-human AILIM mouse monoclonal antibody

FIG. 69 shows the inhibitory effect of various human anti-human AILIMmonoclonal antibodies on the proliferation of T cells in the assay usingmixed lymphocyte reaction (MLR). T cells from a normal healthy person“donor G” were co-cultured with PBMCs from a normal healthy person“donor F” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentrations of test substances.

Other notations are as follows:

“anti-KLH”: human anti-KLH monoclonal antibody as a negative control.

“JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

“138”: human anti-human AILIM monoclonal antibody JMab1 38.

“139”: human anti-human AILIM monoclonal antibody JMab139.

“140”: human anti-human AILIM monoclonal antibody JMab140.

“141”: human anti-human AILIM monoclonal antibody JMab 141.

FIG. 70 shows ADCC-inducing activity of various human anti-human AILIMmonoclonal antibodies and control antibodies where wild-type CHO cellswere used as the target cells.

The vertical axis indicates the rate of cytotoxicity caused byADCC-inducing activity of antibody, and the horizontal axis indicatesthe concentration of antibody.

FIG. 71 shows ADCC-inducing activity of various human anti-human AILIMmonoclonal antibodies and control antibody where humanAILIM-overexpressing recombinant CHO cells were used as the targetcells.

The vertical axis indicates the frequency of cell damage resulted fromthe ADCC-inducing activity of antibody, and the horizontal axisindicates the concentration of antibody.

FIG. 72 shows the inhibitory effect of anti-AILIM antibody on delayedallergy.

The vertical axis indicates the size of redness measured as an index ofthe onset of delayed allergy, and the horizontal axis indicates the typeof test sample given to animal subjects

FIG. 73 shows proliferation activity of monkey T cells in the assay todetermine the activity of various human anti-human AILIM monoclonalantibodies to transduce costimulatory signal, using a microplate coatedwith human anti-human AILIM monoclonal antibody together with anti-humanCD3 monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of the human anti-humanAILIM monoclonal antibody.

In this figure, “anti-KLH” indicates result of assay in which humananti-KLH monoclonal antibody was used as the negative control, insteadof the human anti-human AILIM monoclonal antibody.

FIG. 74 shows inhibitory activity of the negative control antibodyagainst binding between soluble AILIM ligands (hB7h-IgFc) and solubleAILIM (AILIM-IgFc) of various concentrations.

The vertical axis indicates absorbance as an index for the inhibitoryactivity, and the horizontal axis indicates the concentration of solubleAILIM.

FIG. 75 shows inhibitory activity of anti AILIM antibody against bindingbetween soluble AILIM ligands (hB7h-IgFc) and soluble AILIM (AILIM-IgFc)of various concentrations.

The vertical axis indicates absorbance as an index for the inhibitoryactivity, and the horizontal axis indicates the concentration of solubleAILIM.

FIG. 76 shows inhibitory activity of anti AILIM antibody at variousconcentrations against binding between soluble AILIM ligands (hB7h-IgFc)and soluble AILIM (AILIM-IgFc).

The vertical axis indicates absorbance as an index for the inhibitoryactivity, and the horizontal axis indicates the concentration of solubleAILIM.

FIG. 77 shows inhibitory activity of various human anti-human AILIMmonoclonal antibodies to human T cell proliferation in the assay todetermine the activity of transducing costimulatory signal using amicroplate coated with soluble human AILIM ligand (hB7h-IgFc) togetherwith anti-human CD3 monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of antibody.

FIG. 78 shows inhibitory activity of various human anti-human AILIMmonoclonal antibodies to monkey T cell proliferation in the assay todetermine the activity of transducing costimulatory signal using amicroplate coated with soluble human AILIM ligand (hB7h-IgFc) togetherwith anti-human CD3 monoclonal antibody.

The vertical axis indicates the amount of cellular incorporation of[³H]thymidine as an index of the degree of cell proliferation, and thehorizontal axis indicates the concentration of antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions are described in detail herein below by definingterminologies of the present invention.

Herein, “mammal” means human, bovine, goat, rabbit, mouse, rat, hamster,and guinea pig; preferred is human, rabbit, rat, hamster, or mouse andparticularly preferred is human, rat, hamster, or mouse.

The term “mammals other than humans” and “non-human mammals” usedherein, are synomic to each other, meaning all mammals other than humansdefined above.

The term “amino acids” used herein, means every amino acid existing innature, preferably those described according to the alphabetical threeletters system or single letter system as shown below:

glycine (Gly/G), alanine (Ala/A), valine (Val/V), leucine (Leu/L),isoleucine (Ile/I), serine (Ser/S), threonine (Thr/T), aspartic acid(Asp/D), glutamic acid (Glu/E), asparagine (Asn/N), glutamine (Gln/Q),lysine (Lys/K), arginine (Arg/R), cysteine (Cys/C), methionine (Met/M),phenylalanine (Phe/F), tyrosine (Tyr/Y), tryptophan (Trp/W), histidine(His/H), proline (Pro/P).

The term “AILIM” used herein is the abbreviation for ActivationInducible Lymphocyte Immunomodulatory Molecule, indicating a mammaliancell surface molecule having the structure and function as alreadydescribed in a previous report, more preferably a human-derived AILIM inparticular (for example, International Immunology, Vol. 12, No. 1, p.51-55; GenBank Accession Number: BAA82129 (human), BAA82128 (rat),BAA82127 (rat variant), and BAA82126 (mouse)).

Alternatively, this AILIM is also referred to as ICOS (UnexaminedPublished Japanese Patent Application (JP-A) No. Hei 11-29599,International Patent Application No. WO98/38216), and theseabbreviations indicate the same molecule.

“AILIM ligand” used herein means a cell surface molecule which interactswith said co-stimulatory molecule AILIM (ICOS), and is referred to asB7h, B7RP-1, GL50 or LICOS (Nature, Vol. 402, No. 6763, p. 827-832,1999; Nature Medicine, Vol. 5, No. 12, p. 1365-1369, 1999; J.Immunology, Vol. 164, p. 1653-1657, 2000; Curr. Biol. Vol. 10, No. 6, p.333-336, 2000).

Moreover, “AILIM” used herein also includes a polypeptide havingsubstantially the same amino acid sequence as that of AILIM of eachmammal described in the references, and particularly preferably, that ofhuman AILIM. Furthermore, a human AILIM variant which is similar to therat AILIM variant already reported (GenBank Accession Number: BAA82127)is also included in “AILIM” of this invention.

“AILIM ligand” used herein is also defined to have a similar meaning asabove.

Herein, “polypeptides having essentially identical amino acid sequence”means variant polypeptides as described below.

That is, as long as these variant polypeptides have biologicalproperties essentially equivalent to the natural type AILIM(particularly preferably the human-derived AILIM), they are polypeptidesof this invention. Like those having amino acid sequence of the naturaltype AILIM, in which a plurality of amino acid residues, preferably 1 to10 amino acid residues, most preferably 1 to 5 amino acid residues aredeleted and/or modified, and to which a plurality of amino acidresidues, preferably 1 to 10 amino acid residues, most preferably 1 to 5amino acid residues are added.

Furthermore, they may be variant polypeptides having plurality of thesesubstitution, deletion, modification and addition of amino acid residuesin the molecule.

“AILIM ligand” in this invention is also defined to have a similarmeaning as above.

AILIM (particularly human AILIM) and AILIM ligand (particularly humanAILIM ligand) in this invention can be prepared by, in addition to generecombinant technique, appropriately using well-known methods in thistechnical field such as chemical synthesis method, cell culture method,etc. or these methods with modifications.

Such substitution, deletion, or insertion of amino acids can be achievedaccording to the usual method (Experimental Medicine: SUPPLEMENT,“Handbook of Genetic Engineering” (1992); and so on).

Examples of methods for producing mutant polypeptides as mentioned aboveare synthetic oligonucleotide site-directed mutagenesis (gapped duplexmethod), point mutagenesis by which a point mutation is introduced atrandom by treatment with nitrite or sulfite, the method by which adeletion mutant is prepared with Bal31 enzyme and the like, cassettemutagenesis, linker scanning method, miss incorporation method, mismatchprimer method, DNA segment synthesis method, etc.

Synthetic oligonucleotide site-directed mutagenesis (gapped duplexmethod) can be, for example, performed as follows. The region desired tobe mutagenized is cloned into M13 phage vector having amber mutation toprepare the single-stranded phage DNA. After RF I DNA of M13 vectorwithout amber mutation is linearized by restriction enzyme treatment,DNA is mixed with the single-stranded phage DNA mentioned above,denatured, and annealed thereby forming “gapped duplex DNA.” A syntheticoligonucleotide into which mutations are introduced is hybridized withthe gapped duplex DNA and the closed-circular double-stranded DNAs areprepared by the reactions with DNA polymerase and DNA ligase. E. colimutS cells, deficient in mismatch repair activity, are transfected withthis DNA. E. coli cells without suppressor activity are infected withthe grown phages, and only phages without amber mutation are screened.

The method by which a point mutation is introduced with nitriteutilizes, for example the principle as mentioned below. If DNA istreated with nitrite, bases are deaminated to change adenine intohypoxanthine, cytosine into uracil, and guanine into xanthine. Ifdeaminated DNA is introduced into cells, “A:T” and “G:C” are replacedwith “G:C” and “A:T”, respectively, because hypoxanthine, uracil, andxanthine form a base pair with cytosine, adenine, and thymine,respectively, in the DNA replication. Actually, single-stranded DNAfragments treated with nitrite are hybridized with “gapped duplex DNA”,and thereafter mutant strains are separated by manipulating in the sameway as synthetic oligonucleotide site-directed mutagenesis (gappedduplex method).

In addition, “AILIM” herein also includes “a portion” of said AILIM.Herein, “a portion” means a polypeptide comprising any partial sequenceof the above-defined AILIM amino acid sequence.

Preferably, said portion indicates the extracellular region ofabove-defined AILIM (particularly preferably a human AILIM) or anyportion thereof.

“AILIM ligand” in this invention is also defined to have a similarmeaning as above.

“Portion” of said AILIM (preferably the extracellular region of AILIM orany portion thereof) can be prepared according to well-known methods inthis technical field as described below or according to their modifiedmethods by genetic recombination technique or chemical synthesis method,or by suitably cleaving AILIM (particularly preferably a human AILIM)isolated by cell culture method using proteolytic enzymes, etc.

“Portion of AILIM ligand” can be also prepared by similar methods asdescribed above.

“Human antibody” of this invention is a human antibody which binds tothe above-defined AILIM or a portion thereof (particularly preferably ahuman-derived AILIM or a portion thereof). Specifically, it means ahuman-derived polyclonal antibody (human polyclonal antibody, humanantiserum) or human-derived monoclonal antibody (human monoclonalantibody).

“Human monoclonal antibody” of this invention is a human monoclonalantibody which binds to the above-defined AILIM or a portion thereof(particularly preferably a human-derived AILIM or a portion thereof).

More specifically, all the regions comprising the variable and constantregions of the heavy chain (H-chain), and the variable and constantregions of the light chain (L-chain) consist of human immunoglobulinderived from gene encoding said human immunoglobulin. L-chain isexemplified by human K chain or human λ chain.

Human monoclonal antibody which binds to AILIM (particularly preferablya human-derived AILIM) of this invention or a portion thereof is a humanmonoclonal antibody having characteristic defined in any ofaforementioned (5) through (42) or (84).

More specifically, it includes various human monoclonal antibodieshaving various characteristics and industrial applicability as describedin examples and drawings below.

A preferred embodiment of human monoclonal antibody of this invention isa human monoclonal antibody which binds to AILIM or a portion thereofdefined in any of aforementioned (5) through (42) or (84).

Most preferable embodiment is a human monoclonal antibody which binds tohuman AILIM as described in (30) or (39).

“Human monoclonal antibody” of this invention can be prepared byimmunizing following transgenic non-human mammals producing humanantibody with any of the immunogens (antigens) described below.

(a) a natural cell or artificially established cell line expressingaforementioned AILIM (particularly preferably a human-derived AILIM) onthe cell surface;

(b) a genetic recombinant cell prepared using genetic recombinationtechniques so as to express above-defined AILIM (particularly preferablya human-derived AILIM) on the cell surface;

(c) a cell lysate obtained by solubilizing cells aforementioned in (a)or (b), or a polypeptide fragment of AILIM (particularly preferably ahuman-derived AILIM) purified from said cell lysate;

(d) a genetic recombinant cell prepared using genetic recombinationtechniques so as to express a portion (particularly preferably theextracellular region or any preferable peptide thereof) of above-definedAILIM (particularly preferably a human-derived AILIM) as a solublepolypeptide;

(e) a culture supernatant obtained by culturing the genetic recombinantcell aforementioned in (d) or an extracellular region polypeptide(soluble AILIM) of AILIM (particularly preferably a human-derived AILIM)purified from said culture supernatant; or

(f) a portion (particularly preferably the extracellular region or anypreferable peptide thereof) of chemically synthesized AILIM(particularly preferably a human-derived AILIM).

Furthermore, monoclonal antibody of this invention can be also obtainedfrom culture supernatant by culturing a “genetic recombinant host”[herein, said host is an eukaryotic cell other than fertilized eggs(preferably mammalian cells such as CHO, lymphocytes, and myelomacells)], which can be prepared by transforming a host with cDNAs(preferably a vector containing said cDNAs) encoding each of the heavyand light chains of such a human monoclonal antibody of this inventionusing genetic recombination techniques, and which produces geneticrecombinant human monoclonal antibody.

Specifically, the monoclonal antibody of this invention can be obtainedby culturing genetic recombinant host described in any of aforementioned(60) through (62) or (64) through (80) of this invention (herein, saidhost is an eukaryotic cell other than a fertilized egg (preferablymammalian cells such as CHO, lymphocytes, and myeloma cells)).

In addition, human monoclonal antibody of this invention may be a humanmonoclonal antibody having any isotype belonging to IgG (IgG1, IgG2,IgG3 and IgG4), IgH, IgA (IgA1 and IgA2), IgD or IgE. Preferably, saidmonoclonal antibody belongs to IgG (IgG1, IgG2, IgG3 and IgG4), morepreferably IgG1, IgG2 or IgG4.

Human monoclonal antibody of this invention can be prepared byimmunizing transgenic non-human mammal producing human antibody such ashuman antibody-producing transgenic mouse described below with any ofthe immunogens (antigens) aforementioned in (a) through (f) according toknown commonly used manufacturing method.

That is, for example, said transgenic non-human mammal producing humanantibody is immunized with said antigen in combination with Freund'sadjuvant as the occasion demands. Polyclonal antibody can be obtainedfrom sera collected from said immunized animal. Monoclonal antibody canbe manufactured by preparing fusion cells (hybridomas) from saidantibody-producing cells isolated from said immunized animal and myelomacells with no autoantibody-producing ability, and cloning saidhybridomas to select a clone producing the monoclonal antibody with aspecific affinity to the antigen used for immunizing the mammal.

More specifically, monoclonal antibody can be prepared as describedbelow. That is, said human antibody-producing transgenic non-humanmammal (particularly preferably “human antibody-producing transgenicmouse”) is immunized by injecting any of the immunogens aforementionedin (a) through (c) intradermally, intramuscularly, intravenously, intothe footpad, or intraperitoneally once to several times, ortransplanting said immunogen into said mammal. Usually, immunizationsare performed once to four times every one to fourteen days after thefirst immunization. Antibody-producing cells are obtained from themammal so immunized in about one to five days after the lastimmunization. The frequency and interval of immunizations can beappropriately arranged depending on, e.g., property of the immunogenused.

Hybridomas that secrete a human monoclonal antibody can be prepared bythe method of Köhler and Milstein (Nature, Vol. 256, pp. 495-497 (1975))and by its modified method. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from the human antibody-producing transgenicnon-human mammal immunized as mentioned above, preferably a spleen, withmyelomas without autoantibody-producing ability, which are derived from,preferably, a mammal such as a mouse, rat, guinea pig, hamster, rabbit,or human, or more preferably, a mouse, rat, or human.

For example, mouse-derived myeloma P3/X63-AG8.653 (ATCC No. CRL-1580),P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2),NSO, PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., orhuman-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.

Cells producing monoclonal antibodies (for example, hybridomas) can bescreened by cultivating the cells, for example, in microtiter plates andby measuring the reactivity of the culture supernatant in the well inwhich hybridoma growth is observed, to the immunogen used for theimmunization mentioned above, for example, by enzyme immunoassay such asradio immunoassay (RIA) and enzyme-linked immuno-solvent assay (ELISA).

The monoclonal antibodies can be produced from hybridomas by cultivatingthe hybridomas in vitro or in vivo such as in the ascites fluid of amouse, rat, guinea pig, hamster, or rabbit, preferably a mouse or rat,more preferably mouse and isolating the antibodies from the resultingthe culture supernatant or ascites fluid of a mammal.

Monoclonal antibodies of this invention can be manufactured on a largescale by the following method:

(1) genes (cDNAs, etc.) encoding each of the heavy and light chains ofsaid monoclonal antibody are cloned from said hybridomas;

(2) cloned genes encoding each of the heavy and light chains areinserted into separate vectors or a single vector to prepare theexpression vector;

(3) said expression vector is transferred into a fertilized egg of adesired non-human mammal (such as goat);

(4) said fertilized egg transferred with the gene is transplanted intothe uterus of a foster mother to obtain a chimeric non-human animal;

(5) by further mating said chimeric goat with another non-human mammal,a transgenic non-human mammal (cattle, goat, sheep or swine) with genesencoding each of said heavy and light chains incorporated into theendogenous gene is produced; and

(6) from the milk of said transgenic non-human mammal, monoclonalantibody derived from said human monoclonal antibody gene is obtained ona large scale (Nikkei Science, April, 1997, p. 78-84).

Cultivating in vitro the cells producing the monoclonal antibodies canbe performed depending on, e.g., the property of cells to be cultured,the object of a test study, and the various conditions of a cultivatingmethod, by using known nutrient media or any nutrient media derived fromknown basal media for growing, maintaining, and storing the hybridomasto produce monoclonal antibodies in culture supernatant.

Examples of basal media are low calcium concentration media such asHam′F12 medium, MCDB153 medium, or low calcium concentration MEM medium,and high calcium concentration media such as MCDB104 medium, MEM medium,D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basalmedia can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

Monoclonal antibodies can be isolated and purified from the culturesupernatant or ascites fluid mentioned above by saturated ammoniumsulfate precipitation, euglobulin precipitation method, caproic acidmethod, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

Human monoclonal antibody of this invention includes human monoclonalantibodies consisting of the heavy chain and/or light chain of whichamino acid sequence for each chain have one or more amino acid residuesdeleted, substituted or added.

Herein, “more amino acid residues” means a plurality of amino acids,specifically 1 to 10 amino acid residues, preferably 1 to 5 amino acidresidues.

A partial modification (deletion, substitution, insertion or addition)as described above can be introduced into the amino acid sequence ofhuman monoclonal antibody of this invention by partial alteration ofbase sequence encoding said amino acid sequence. This partial alterationof base sequence can be introduced by standard method using knownsite-specific mutagenesis technique (Proc. Natl. Acad. Sci. USA, Vol.81, p. 5662-5666, 1984).

“Transgenic human antibody-producing non-human mammal”, particularlyhuman antibody-producing transgenic mouse which is a preferredembodiment, can be prepared according to published literature (NatureGenetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156,1997; Published Japanese Translation of International Publication No.Hei 4-504365; Published Japanese Translation of Publication No. Hei7-509137; Nikkei Science, June, p. 40-50, 1995; International PatentPublication No. WO94/25585; Nature, Vol. 368, p. 856-859, 1994; andPublished Japanese Translation of Publication No. Hei 6-500233, etc.)

Specifically, said human antibody-producing transgenic mice can beprepared, for example, using techniques consisting of the followingprocesses:

(1) preparing a knockout mouse which endogenous immunoglobulin heavychain gene is functionally inactivated by substituting at least aportion of gene locus of the mouse endogenous immunoglobulin heavy chainwith a drug tolerance marker gene (such as neomycin tolerance gene) byhomologous recombination;

(2) preparing a knockout mouse which endogenous immunoglobulin lightchain gene (particularly the κ chain gene) is functionally inactivatedby substituting at least a portion of gene locus of the mouse endogenousimmunoglobulin light chain with a drug tolerance marker gene (such asneomycin tolerance gene) by homologous recombination;

(3) preparing a transgenic mouse which desired region of the humanimmunoglobulin heavy chain gene locus is incorporated into the mousechromosome using a vector represented by the yeast artificial chromosome(YAC) capable of carrying a giant gene;

(4) preparing a transgenic mouse which desired region of the humanimmunoglobulin light chain gene locus (particularly the K chain gene) isincorporated into the mouse chromosome using a vector represented by theyeast artificial chromosome (YAC) capable of carrying a giant gene; and

(5) preparing a transgenic mouse which endogenous immunoglobulin heavyand light chains gene loci are both functionally inactivated and whichchromosome is incorporated with the desired regions of both of the humanimmunoglobulin heavy and light chains gene loci by mating knockout andtransgenic mice aforementioned in (1) through (4) in arbitrary orders.

The above-described knockout mouse can be prepared by substituting thesuitable region of the mouse endogenous immunoglobulin gene locus with aforeign marker gene (such as neomycin tolerance gene) based onhomologous recombination to inactivate said gene locus so as not to berearranged. For the inactivation using said homologous recombination,for example, a method referred to as positive negative selection (PNS)can be used (Nikkei Science, May, p. 52-62, 1994).

Functional inactivation of the immunoglobulin heavy chain gene locus canbe achieved, for example, by introducing a lesion into a part of the J-or C-region (for example, Cμ region). And functional inactivation of theimmunoglobulin light chain (for example, κ chain) can be achieved, forexample, by introducing a lesion into a part of J- or C-region, or aregion extending over J- and C-regions.

A transgenic mouse can be prepared according to the method as usuallyused for producing a transgenic animal (for example, see “Newest Manualof Animal Cell Experiment”, LIC press, Chapter 7, pp. 361-408, (1990)).Specifically, for example, the HPRT-negative (hypoxanthine-guaninephosphoribosyltransferase gene deficient) ES cell (embryonic stem cell)derived from a normal mouse blastocyst is fused with yeast containingthe YAC vector inserted with the gene encoding said human immunoglobulinheavy chain gene locus or light chain gene locus or a portion thereofand the HPRT gene using spheroplast fusion method. ES cells whose mouseendogenous gene is integrated with said foreign gene are selected by HATselection method. Then, the ES cells screened are microinjected into afertilized egg obtained from another normal mouse (blastocyst) (Proc.Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384 (1980); U.S. Pat.No. 4,873,191). The blastocyst is transplanted into the uterus ofanother normal mouse as the foster mother. Then, chimeric transgenicmice are born from the foster mother mouse. By mating the chimerictransgenic mice with normal mice, heterogeneic transgenic mice areobtained. By mating the heterogeneic transgenic mice with each other,homogeneic transgenic mice are obtained according to Mendel's laws.

The “portion of a monoclonal antibody” used in the present inventionmeans a partial region of the above-mentioned human monoclonal antibodyof the present invention, and specifically, includes F(ab′)₂, Fab′, Fab,Fv (variable fragment of antibody), sFv, dsFv (disulfide stabilized Fv),or dAb (single domain antibody) (Exp. Opin. Ther. Patents, Vol. 6, No.5, pp. 441-456 (1996)).

“F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds in the hinge regions existing between each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds in the hinge regions existing between each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof V_(L) (L chain variable region) and C_(L) (L chain constant region),and an H chain fragment composed of V_(H) (H chain variable region) andC_(H)γ1 (γ1 region in the constant region of H chain) are connected attheir C terminal regions through a disulfide bond. Each of such twohomologous antibody fragments is called Fab′. Pepsin also cleaves IgGdownstream of the disulfide bonds in the hinge regions existing betweeneach of the two H chains to generate an antibody fragment slightlylarger than the fragment in which the two above-mentioned Fab′ areconnected at the hinge region. This antibody fragment is called F(ab′)₂.

“Binding rate constant (ka)” herein means a value indicating the bindingstrength (degree) of said monoclonal antibody to the target antigencalculated based on the antibody antigen reaction kinetics.“Dissociation rate constant (kd)” means a value indicating thedissociation strength (degree) of said monoclonal antibody from thetarget antigen. “Dissociation constant (Kd)” is a value obtained bydividing said “dissociation rate constant (kd)” by said “binding rateconstant (ka)” value. These constants are used to represent the affinityof said monoclonal antibody to antigen and its activity to neutralizeantigen.

Said constants can be analyzed according to various methods, and can beeasily analyzed using a commercial assay kit BiacoreX (AmershamPharmacia) or a similar kit according to the manual and experimentalmethod attached to said kit. ka, kd and Kd values obtained using saidkit are expressed in 1/M.Sec, 1/Sec and M (mol) units, respectively.Higher ka values indicate stronger antigen binding activity ofmonoclonal antibody tested, and smaller Kd values show stronger antigenneutralizing activity of antibody.

Human monoclonal antibody of this invention includes those having theka, kd or Kd value as shown in following (1) through (3):

(1) human monoclonal antibody which binds to human AILIM or a portionthereof with the binding rate constant (ka) of 1.0×10⁴ (1/M.Sec) ormore, preferably 1.0×10⁵ (1/M.Sec) or more.

(2) human monoclonal antibody which binds to human AILIM or a portionthereof with the dissociation rate constant (kd) of 1.0×10⁻⁴ (1/Sec) orless, preferably 1.0×10⁻⁵ (1/Sec) or less.

(3) human monoclonal antibody which has a reactivity to human AILIM or aportion thereof with the dissociation constant (Kd) of 1.0×10⁻⁷ (M) orless, preferably 1.0×10⁻⁸ (M) or less and more preferably 1.0×10⁻⁹ (M)or less.

In this case, each value of ka, kd and Kd described above is expected toslightly fluctuate depending on various conditions at the time ofmeasurement with a margin of error but with practically no fluctuationin indexes in general.

“Monoclonal antibody-producing cell” or genetic recombinant humanmonoclonal antibody-producing “genetic recombinant host” of thisinvention (herein, said host is a cell excluding fertilized egg) meansany cell producing aforementioned human monoclonal antibody of thisinvention.

Specifically, for example, it includes cells described in any offollowing (1) through (3), but is not limited to them:

(1) human monoclonal antibody-producing B cell obtained by immunizingaforementioned human antibody-producing transgenic non-human mammal withthe above-defined immunogen (antigen) and collecting the cell from saidimmunized animal.

(2) aforementioned fusion cell (hybridoma) resulted by fusion of thehuman monoclonal antibody-producing B cell thus obtained with a myelomacell derived from mammal.

(3) genetic recombinant human monoclonal antibody-producing geneticrecombinant cell obtained by transforming a cell excluding said B celland hybridoma (for example, CHO (chinese hamster ovarian) cell, BHK(baby hamster kidney) cell, lymphocyte such as myeloma) with the geneencoding said human monoclonal antibody (gene encoding the heavy chainor that encoding the light chain, or both genes) isolated from saidhuman monoclonal antibody-producing B cell or human monoclonalantibody-producing fusion cell (hybridoma).

Herein, the genetic recombinant human monoclonal antibody-producinggenetic recombinant cell aforementioned in (3) namely means a geneticrecombinant cell producing the genetic recombinant of human monoclonalantibody generated by the B cell described above in (1) or the hybridomaaforementioned in (2).

And, “host” in “genetic recombinant host” of this invention includes, inaddition to various mammalian cells as described above, fertilized eggsof any non-human mammals (goat, swine, sheep, cattle, etc.). Bytransferring a gene (gene encoding the heavy chain or that encoding thelight chain, or both genes) encoding any monoclonal antibody (preferablyhuman monoclonal antibody) to human AILIM of this invention into thisfertilized egg, a genetic recombinant fertilized egg of this inventioncan be obtained. This genetic recombinant fertilized egg is used toprepare transgenic animals for manufacturing the aforementioned proteinfrom the milk on a large scale (Nikkei Science, April, 1997, p. 78-84).

“A substance” composing the present invention, specifically “a substancehaving an activity in modulating the signal transduction mediated byAILIM”, and more specifically “a substance having an activity ininhibiting proliferation of AILIM-expressing cells, or in inhibitingproduction of a cytokine by AILIM-expressing cells” means a naturalsubstance present in the nature, or a artificially prepared arbitrarysubstance.

“Substance” related to “substance binding to AILIM” and “substancebinding to AILIM ligand” herein also means any natural substance innature or any artificially prepared substance.

Here, “the signal transduction mediated by AILIM” means the signaltransduction through AILIM, leading to a change of an arbitraryphenotype in the AILIM-expressing cells (cell proliferation, activationof cells, inactivation of cells, apoptosis, and/or a change of anability for producing an arbitrary cytokine from AILIM-expressingcells).

“The substance” can be mainly classified into “a protein substance” and“a non-protein substance”.

Examples of the “protein substances” are the following polypeptide,antibody (a polyclonal antibody, a monoclonal antibody, or a portion ofa monoclonal antibody, and particularly preferably the human antibodymentioned above).

When the substance is an antibody, the substance is preferably amonoclonal antibody. When the substance is a monoclonal antibody, thesubstance includes not only a non-human mammal derived monoclonalantibody, but also a recombinant chimeric monoclonal antibody, arecombinant humanized monoclonal antibody and human monoclonal antibody.

Here, the “recombinant chimeric monoclonal antibody” is a monoclonalantibody prepared by genetic engineering, and specifically means achimeric antibody such as mouse/human chimeric monoclonal antibody whosevariable regions are derived from immunoglobulin of an non-human mammal(mouse, rat, hamster, etc.) and whose constant regions are derived fromhuman immunoglobulin.

The “humanized monoclonal antibody (CDR-grafted antibody)” of thepresent invention is a monoclonal antibody prepared by geneticengineering and specifically means a humanized monoclonal antibodywherein a portion or the whole of the complementarity determiningregions of the hypervariable region are derived from the complementaritydetermining regions of the hypervariable region from a monoclonalantibody of an non-human mammal (mouse, rat, hamster, etc.), theframework regions of the variable region are derived from the frameworkregions of the variable region from human immunoglobulin, and theconstant region is derived from human a constant region fromimmunoglobulin.

The complementarity determining regions of the hypervariable regionexists in the hypervariable region in the variable region of an antibodyand means three regions which directly and complementary binds to anantigen (complementarity-determining residues, CDR1, CDR2, and CDR3).The framework regions of the variable region mean four comparativelyconserved regions lying upstream, downstream or between the threecomplementarity determining regions (framework region, FR1, FR2, FR3,and FR4).

In other words, a humanized monoclonal antibody means that in which allthe regions except a portion or the whole of the complementaritydetermining regions of the hypervariable region of a non-humanmammal-derived monoclonal antibody have been replaced with theircorresponding regions derived from a human immunoglobulin.

The constant region derived from human immunoglobulin has the amino acidsequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonalantibody in the present invention can be that from human immunoglobulinbelonging to any isotype. Preferably, it is the constant region of humanIgG. The framework regions of the constant region derived from humanimmunoglobulin are not particularly limited.

When the substance of the present invention is a polypeptide, thesubstance includes the following polypeptide, a fragment of thepolypeptide (an oligopeptide), a fusion polypeptide, a chemicallymodified one thereof. Examples of an oligopeptide are a peptidecomprising 5 to 30 amino acids, preferably 5 to 20 amino acids. Thechemical modification can be designed depending on various purposes, forexample, the increased half-life in blood in the case of administeringin vivo, or the increased tolerance against the degradation or increasedabsorption in digestive tract at the oral administration.

Examples of the polypeptide are as follows:

-   (1) A polypeptide comprising the whole or a portion of an    extracellular region of AILIM;-   (2) A fusion polypeptide comprising the whole or a portion of an    extracellular region of AILIM and the whole or a portion of a    constant region of immunoglobulin heavy chain; or-   (3) A polypeptide which binds to AILIM.

Examples of the “non-protein” are DNA, RNA, and a chemically synthesizedcompound.

Here, “DNA” means “DNA comprising a partial nucleotide sequence of theDNA or chemically modified DNA thereof” useful as an antisense DNApharmaceutical designed based on a nucleotide sequence of DNA (includingcDNA and genomic DNA) encoding the above AILIM (preferably human AILIM).Specifically the antisense DNA can inhibit transcription of DNA encodingthe AILIM into mRNA, or translation of the mRNA into a protein byhybridizing DNA or RNA encoding AILIM.

The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides in an arbitrary region. The partial nucleotide sequenceconsists of 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and still more preferably 5 to 30 consecutive nucleotides.

When the DNA is used as an antisense DNA pharmaceutical, the DNAsequence can be modified chemically in part for extending the half-life(stability) of the blood concentration of the DNA administered topatients, for increasing the intracytoplasmic-membrane permeability ofthe DNA, or for increasing the degradation resistance or the absorptionof the orally administered DNA in the digestive organs. The chemicalmodification includes, for example, the modification of the phosphatebonds, the riboses, the nucleotide bases, the sugar moiety, the 3′ endand/or the 5′ end in the structure of the oligonucleotide DNA.

The modification of phosphate bond includes, for example, the conversionof one or more of the bonds to phosphodiester bonds (D-oligo),phosphorothioate bonds, phosphorodithioate bonds (S-oligo), methylphosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds ormethyl phosphonothioate bonds, or combinations thereof. The modificationof the ribose includes, for example, the conversion to 2′-fluororiboseor 2′-O-methylribose. The modification of the nucleotide base includes,for example, the conversion to 5-propynyluracil or 2-aminoadenine.

Here, “RNA” means “RNA comprising a partial nucleotide sequence of theRNA or chemically modified RNA thereof” useful as an antisense RNApharmaceutical designed based on a nucleotide sequence of RNA encodingthe above AILIM (preferably human AILIM). The antisense RNA can inhibittranscription of DNA encoding the AILIM into mRNA, or translation of themRNA into a protein by hybridizing DNA or RNA encoding AILIM.

The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides in an arbitrary region. The partial nucleotide sequenceconsists of 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and still more preferably 5 to 30 consecutive nucleotides.

The sequence of antisense RNA can be modified chemically in part forextending the half-life (stability) of the blood concentration of theRNA administered to patients, for increasing theintracytoplasmic-membrane permeability of the RNA, or for increasing thedegradation resistance or the absorption of the orally administered RNAin the digestive organ. An example of chemical modification is thechemical modification applied to the above antisense DNA.

Examples of “a chemically synthesized compound” are an arbitrarycompound except for the above DNA, RNA and protein substances, havingthe molecular weight of about 100 to about 1000, preferably a compoundhaving the molecular weight of about 100 to about 800, and morepreferably the molecular weight of about 100 to about 600.

A “polypeptide” included in the definition of the above “substance”means a portion (a fragment) of a polypeptide chain constituting AILIM(preferably human AILIM), preferably the whole or a portion of anextracellular region of the polypeptide constituting AILIM (1 to 5 aminoacids may be optionally added into the N-terminus and/or C-terminus ofthe region).

AILIM involving in the present invention is a transmembrane moleculepenetrating cell membrane, comprising 1 or 2 polypeptide chains.

Here, a “transmembrane protein” means a protein that connects withmembrane through the hydrophobic peptide region penetrating the lipidbilayer of the membrane once or several times and whose structure is, asa whole, composed of three main regions, that is, extracellular region,transmembrane region, and cytoplasmic region, as seen in many receptorsor cell surface molecules. Such a transmembrane protein constitutes eachreceptor or cell surface molecule in the form of a monomer, homodimer,heterodimer or oligomer with another chain(s) having the same ordifferent amino acid sequence.

Here, an “extracellular region” means the whole or a portion from thepartial structure (partial region) from the entire structure of theabove-mentioned transmembrane protein where the partial structure existsoutside of the membrane. In other words, it means the whole or a portionof the region of the transmembrane protein except the regionincorporated into the membrane (transmembrane region) and the regionexisting in the cytoplasm following the transmembrane region(cytoplasmic region).

“A fusion polypeptide” included in the above “protein substance” means afusion polypeptide comprising the whole or a portion of an extracellularregion of a polypeptide constituting AILIM (preferably human AILIM), and“the whole or a portion of a constant region of immunoglobulin heavychain (Ig, preferably human Ig)”. Preferably, the fusion polypeptide isa fusion polypeptide with an extracellular region of AILIM and a portionof a constant region of human IgG heavy chain and particularlypreferably, a fusion polypeptide of an extracellular region of AILIM anda region (Fc) of human IgG heavy chain comprising a hinge region, CH2domain and CH3 domain. As IgG, IgG1 is preferable, and as AILIM, human,mouse, or rat AILIM is preferable (preferably human).

“The whole or a portion of a constant region of human immunoglobulin(Ig) heavy chain” used herein means the constant region or the Fc regionof human-derived immunoglobulin heavy chain (H chain) as described, or aportion thereof. The immunoglobulin can be any immunoglobulin belongingto any class and any subclass. Specifically, examples of theimmunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 andIgA2), IgD, and IgE. Preferably, the immunoglobulin is IgG (IgG1, IgG2,IgG3, or IgG4), or IgM. Examples of particularly preferableimmunoglobulin of the present invention are those belonging tohuman-derived IgG (IgG1, IgG2, IgG3, or IgG4).

Immunoglobulin has a Y-shaped structural unit in which four chainscomposed of two homologous light chains (L chains) and two homologousheavy chains (H chains) are connected through disulfide bonds (S-Sbonds). The light chain is composed of the light chain variable region(V_(L)) and the light chain constant region (C_(L)). The heavy chain iscomposed of the heavy chain variable region (V_(H)) and the heavy chainconstant region (C_(H)).

The heavy chain constant region is composed of some domains having theamino acid sequences inherent in each class (IgG, IgM, IgA, IgD, andIgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2).

The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composed of VH,CH1 domain, hinge region, CH2 domain, and CH3 domain in this order fromN terminus.

Similarly, the heavy chain of IgG1 is composed of V_(H), CΓ₁1 domain,hinge region, Cγ₁2 domain, and Cγ₁3 domain in this order from Nterminus. The heavy chain of IgG2 is composed of V_(H), Cγ₂1 domain,hinge region, Cγ₂2 domain, and Cγ₂3 domain in this order from Nterminus. The heavy chain of IgG3 is composed of V_(H), Cγ₃1 domain,hinge region, Cγ₃2 domain, and Cγ₃3 domain in this order from Nterminus. The heavy chain of IgG4 is composed of V_(H), Cγ₄1 domain,hinge region, Cγ₄2 domain, and Cγ₄3 domain in this order from Nterminus.

The heavy chain of IgA is composed of V_(H), Cα1 domain, hinge region,Cα2 domain, and Cα3 domain in this order from N terminus.

Similarly, the heavy chain of IgA1 is composed of V_(H), Cα₁1 domain,hinge region, Cα₁2 domain, and Cα₁3 domain in this order from Nterminus. The heavy chain of IgA2 is composed of V_(H), Cα₂1 domain,hinge region, Cα₂2 domain, and Cα₂3 domain in this order from Nterminus.

The heavy chain of IgD is composed of V_(H), Cδ1 domain, hinge region,Cδ2 domain, and Cδ3 domain in this order from N terminus.

The heavy chain of IgM is composed of V_(H), Cμ1 domain, Cμ2 domain, Cμ3domain, and Cμ4 domain in this order from N terminus and have no hingeregion as seen in IgG, IgA, and IgD.

The heavy chain of IgE is composed of V_(H), Cε1 domain, Cε2 domain, Cε3domain, and Cε4 domain in this order from N terminus and have no hingeregion as seen in IgG, IgA, and IgD.

If, for example, IgG is treated with papain, it is cleaved at theslightly N terminal side beyond the disulfide bonds existing in thehinge region where the disulfide bonds connect the two heavy chains togenerate two homologous Fab, in which a heavy chain fragment composed ofV_(H) and CH1 is connected with one light chain through a disulfidebond, and one Fc, in which two homologous heavy chain fragments composedof the hinge region, CH2 domain, and CH3 domain are connected throughdisulfide bonds (See “Immunology Illustrated”, original 2nd ed.,Nankodo, pp. 65-75 (1992); and “Focus of Newest Medical Science‘Recognition Mechanism of Immune System’”, Nankodo, pp. 4-7 (1991); andso on).

Namely, “a portion of a constant region of immunoglobulin heavy chain”mentioned above means a portion of a constant region of animmunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is the constant region without C1domain, or the Fc region. Specifically, example thereof is the regioncomposed of hinge region, C2 domain, and C3 domain from each of IgG,IgA, and IgD, and is the region composed of C2 domain, C3 domain, and C4domain from each of IgM and IgE. A particularly preferable examplethereof is the Fc region of human-derived IgG1.

The fusion polypeptide mentioned above has the advantage that the fusionpolypeptide can be purified extremely easily by using affinity columnchromatography using the property of protein A, which binds specificallyto the immunoglobulin fragment because the fusion polypeptide of thepresent invention has a portion of a constant region (for example Fc) ofan immunoglobulin such as IgG as mentioned above as a fusion partner.Moreover, since various antibodies against the Fc of variousimmunoglobulins are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

“A polypeptide which binds to AILIM” is included in “a polypeptide”included in the definition of the above “substance”.

Specific examples of “a polypeptide which binds to AILIM” are the wholeor a portion of a polypeptide constituting known B7h, B7RP-1, GL50 or amolecule called LICOS which are ligands interacting with AILIM (Nature,Vol. 402, No. 6763, pp. 827-832, 1999; Nature Medicine, Vol. 5, No. 12,pp. 1365-1369, 1999; J. Immunology, Vol. 164, pp. 1653-1657, 2000; Curr.Biol., Vol. 10 No 6, pp. 333-336, 2000).

Preferably, the polypeptide is a polypeptide comprising the whole or aportion of an extracellular region of the above ligands (B7h, B7RP-1,GL50, LICOS), or a fusion polypeptide comprising the polypeptide, andthe whole or a portion of a constant region of immunoglobulin heavychain (preferably human immunoglobulin). Here, the terms “anextracellular region” and “a constant region of immunoglobulin heavychain” have the same meaning as the above.

The polypeptide, a portion of the polypeptide (fragment), and fusionpolypeptide mentioned above can be produced not only by recombinant DNAtechnology as mentioned below but also by a method well known in the artsuch as a chemical synthetic method and a cell culture method, or amodified method thereof.

The “antibody” of the present invention can be a polyclonal antibody(antiserum) or a monoclonal antibody against mammalian AILIM(particularly preferably human AILIM) defined above, and preferably amonoclonal antibody.

Specifically the antibody is an antibody having an activity ininhibiting proliferation of AILIM-expressing cells by biding to AILIM,or inhibiting production of interferon γ or interleukin 4 byAILIM-expressing cells through biding to AILIM.

“Delayed type allergy” herein this allergy mediated by cellular immunity(particularly mediated by Th1-type T cell), that is, the allergy ismediated by T cell sensitized with antigen (memory T cell memorizingantigen) and is referred to any allergy, which takes approximately 24 to48 hours to exhibit allergic reaction accompanied with inflammationcaused by said memory T cell when the living organism sensitized with anantigen is re-contacted with the same antigen.

This delayed type allergy includes allergy to an infectious pathogenicantigen such as tuberculin allergy derived from Mycobacteriumtuberculosis, a transient Jones-Mote delayed type allergy to a minutequantity of protein, contact allergy to chemicals such as picrylchloride or plant toxin such as lacquer, or allergy related to graftrejection to graft observed in the allograft.

“Pharmaceutical composition” herein means a composition useful as a drugcomprising as the effective ingredients antibody (preferably humanantibody), which binds to AILIM (preferably human AILIM) or a portionthereof, or monoclonal antibody (preferably human monoclonal antibody)or a portion thereof and a “pharmacologically acceptable carrier”.

The “pharmaceutically acceptable carrier” includes a excipient, adiluent, an expander, a decomposition agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a solubilityincreasing agent, or other additives.

Using one or more of such carriers, a pharmaceutical composition can beformulated into tablets, pills, powders, granules, injections,solutions, capsules, troches, elixirs, suspensions, emulsions, orsyrups.

The pharmaceutical composition can be administered orally orparenterally. Other forms for parenteral administration include asolution for external application, suppository for rectaladministration, and pessary, prescribed by the usual method, whichcomprises one or more active ingredient.

The dosage can vary depending on the age, sex, weight, and symptom of apatient, effect of treatment, administration route, period of treatment,or the kind of active ingredient (polypeptide or antibody mentionedabove) contained in the pharmaceutical composition. Usually, thepharmaceutical composition can be administered to an adult in a dose of10 μg to 1000 mg (or 10 μg to 500 mg) per one administration. Dependingon various conditions, the dosage less than that mentioned above may besufficient in some cases, and the dosage more than that mentioned abovemay be necessary in other cases.

In particular, the injection can be produced by dissolving or suspendingthe antibody in a non-toxic, pharmaceutically acceptable carrier such asphysiological saline or commercially available distilled water forinjection with adjusting a concentration to 0.1 μg antibody/ml carrierto 10 mg antibody/ml carrier.

The injection thus produced can be administered to a human patient inneed of treatment in a dose of 1 μg to 100 mg/kg body weight, preferably50 μg to 50 mg/kg body weight once or more times a day. Examples ofadministration route are medically appropriate administration routessuch as intravenous injection, subcutaneous injection, intradermalinjection, intramuscular injection, or intraperitoneal injection,preferably intravenous injection.

The injection can also be prepared into a non-aqueous diluent (forexample, propylene glycol, polyethylene glycol, vegetable oil such asolive oil, and alcohol such as ethanol), suspension, or emulsion.

The injection can be sterilized by filtration with abacteria-non-penetrated filter, by mixing bacteriocide, or byirradiation. The injection can be produced in the form that is preparedupon use. Namely, it is freeze-dried to be a sterile solid composition,and can be dissolved in sterile distilled water for injection or anothersolvent before use.

Pharmaceutical compositions comprising the human antibodies of thisinvention are useful as pharmaceutical preparations, without inducinghost immunorejection due to HAMA (human anti-mouse antibody), to controla variety of biological reactions (e.g., proliferation of cellsexpressing AILIM, cytokine production by cells expressing AILIM, immunecytolysis or death (apoptosis) of cells expressing AILIM and others)that are associated with AILIM-mediated transduction of costimulatorysignal (secondary signal) to AILIM-expressing cells, and/or aspharmaceutical preparations to treat or prevent various diseases bysuppressing and inhibiting the onset and/or progress of diseasesassociated with AILIM-mediated signal transduction.

The term “immune cytolysis” herein indicates a biological phenomenon asfollows.

Lysis of the cell (cytolysis) can be induced by an antibody(particularly cell-lysing antibody) as well as by binding with killercells. The cell-lysing antibody is a cytotoxic antibody, whichparticularly has lysing activity on cells such as immune cells, tissuecell or sperms. When the antibody binds to the cell-surface antigen, itcauses a cytotoxic effect on the cell or induces cytolysis in thepresence of the complement.

This immune cytolysis is induced by the action of the complement inconjunction with specific binding of the antibody to cell-surfaceantigen. The antibody bound to the surface antigen activates C1complement (C1). Subsequently cell damage sites are formed through aseries of complement-fixation reactions with C2 to C9 complements(C2-C9), and then cellular contents are released from the cells therebylysing the cells.

The term “antibody-dependent cellular cytotoxicity” herein indicates abiological event that is also abbreviated as “ADCC,” and is a cytotoxicaction on target cells by effector cells such as lymphocytes,macrophages or polymorphonuclear leucocytes, which requires not onlyeffector cells and target cells but also an antibody participating inthe induction of the cytotoxic event.

The term “mixed lymphocyte reaction” herein means a biologicalphenomenon abbreviated as “MLR.” The reaction is also referred to asmixed leukocyte reaction.

Allogenic leukocytes or lymphocytes derived from distinct individualsare mixed with each other and cultured for several days, therebyallowing blast formation of the cells and DNA synthesis in the cells(i.e., cell proliferation). This reaction is referred to as MLR(allogenic MLR).

DNA synthesis (cell proliferation) can be analyzed by arrestingproliferation of either of the lymphocytes. The arrest can beaccomplished by treatment with irradiation or mitomycin. Analysis can becarried out by measuring the amount of DNA synthesized in the otherlymphocyte.

The amount of synthesized DNA can be analyzed by measuring incorporationof thymidine, labeled with radioisotope such as tritium, into thenucleus of the cell.

DNA encoding AILIM (particularly preferably human AILIM) of the presentinvention can be obtained according to a commonly used method by usingprocedures for cloning cDNA from mRNA encoding AILIM; procedure forisolating genomic DNA and splicing them; procedure for preparing the DNAby PCR using a cDNA sequence or mRNA sequence as a template; orprocedure for chemically synthesizing the DNA.

DNA encoding the AILIM ligand in accordance with the present inventioncan also be obtained in the same manner as described above.

DNA encoding AILIM (particularly preferably the human AILIM) of thepresent invention can be prepared by cutting (digesting) DNA comprisingDNA encoding AILIM prepared as such with appropriate restrictionenzymes, and as required, ligating the resultant DNA fragment with alinker DNA or tag by using an appropriate DNA polymerase or the like.The DNA encoding AILIM ligand can also be prepared in the same manner.

An exemplary method will be shown below to clone the cDNA encoding AILIM(particularly preferably the human AILIM; the protein is hereinafterreferred to as the protein of interest) from the mRNA.

A DNA encoding AILIM ligand can also be cloned in the same manner.

First, messenger RNA encoding the protein of interest is prepared fromtissues or cells expressing and producing the protein of interest. mRNAcan be prepared isolating total RNA by a known method such asquanidine-thiocyanate method (Chirgwin, J. M. et al., Biochemistry, Vol.18, p 5294, 1979), hot phenol method, or AGPC method, and subjecting itto affinity chromatography using oligo-dT cellulose or poly-U Sepharose.

Then, with the mRNA obtained as a template, cDNA is synthesized, forexample, by a well-known method using reverse transcriptase such as themethod of Okayama et al. (Mol. Cell. Biol. Vol. 2, p. 161 (1982); ibid.Vol. 3, p. 280 (1983)) or the method of Hoffman et al. (Gene Vol. 25, p.263 (1983)), and converted into double-stranded cDNA. A cDNA library isprepared by transforming E. coli with plasmid vectors, phage vectors, orcosmid vectors having this cDNA or by transfecting E. coli after invitro packaging.

The plasmid vectors used in this invention are not limited as long asthey are replicated and maintained in hosts. Any phage vectors that canbe replicated in hosts can also be used. Examples of usually usedcloning vectors are pUC19, λgt10, λgt11, and so on. When the vector isapplied to immunological screening as mentioned below, the vector havinga promoter that can express a gene encoding the polypeptide of thepresent invention in a host is preferably used.

cDNA can be inserted into a plasmid by, for example, the method ofManiatis et al. (Molecular Cloning, A Laboratory Manual, second edition,Cold Spring Harbor Laboratory, p. 1.53, 1989). cDNA can be inserted intoa phage vector by, for example, the method of Hyunh et al. (DNA cloning,a practical approach, Vol. 1, p. 49 (1985)). These methods can be simplyperformed by using a commercially available cloning kit (for example, aproduct from Takara Shuzo). The recombinant plasmid or phage vector thusobtained is introduced into appropriate host cells such as a prokaryote(for example, E. coli: XL1Blue MRF′, DH5α, HB101, MC1061/P3, etc.).

Examples of a method for introducing a plasmid into a host are calciumchloride method, calcium chloride/rubidium chloride method described inMolecular Cloning, A Laboratory Manual (second edition, Cold SpringHarbor Laboratory, p. 1.74 (1989)), and electroporation method. Phagevectors can be introduced into host cells by, for example, a method inwhich the phage DNAs are introduced into grown hosts after in vitropackaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

The cDNA encoding the polypeptide of the present invention can beisolated from the cDNA library so prepared according to the methodmentioned above by combining general cDNA screening methods.

For example, a clone comprising the desired cDNA can be screened by aknown colony hybridization method (Crunstein et al. Proc. Natl. Acad.Sci. USA, Vol. 72, p. 3961 (1975)) or plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, p. 2.108 (1989)) using ³²P-labeled chemicallysynthesized oligonucleotides as probes, which are corresponding to theamino acid sequence of the polypeptide of the present invention.Alternatively, a clone having a DNA fragment encoding a specific regionwithin the polypeptide of the present invention can be screened byamplifying the region by PCR with synthetic PCR primers.

When a cDNA library prepared using a cDNA expression vector (forexample, λZAPII phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against thepolypeptide of the present invention. A screening method using PCRmethod is preferably used when many clones are subjected to screening.

The nucleotide sequence of the DNA thus obtained can be determined byMaxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, Vol. 74,p. 560 (1977)) or the dideoxynucleotide synthetic chain terminationmethod using phage M13 (Sanger et al. Proc. Natl. Acad. Sci. USA, Vol.74, pp. 5463-5467 (1977)). The whole or a portion of the gene encodingthe polypeptide of the present invention can be obtained by excising theclone obtained as mentioned above with restriction enzymes and so on.

The DNA encoding the polypeptide of the present invention can beisolated from the genomic DNA derived from the cells expressing thepolypeptide of the present invention as mentioned above by the followingmethods.

Such cells are solubilized preferably by SDS or proteinase K, and theDNAs are deproteinized by repeating phenol extraction. RNAs are digestedpreferably with ribonuclease. The DNAs obtained are partially digestedwith appropriate restriction enzymes, and the DNA fragments obtained areamplified with appropriate phage or cosmid to generate a library. Then,clones having the desired sequence are detected, for example, by usingradioactively labeled DNA probes, and the whole or a portion of the geneencoding the polypeptide of the present invention is obtained from theclones by excision with restriction enzyme and so on.

Preparation of DNA encoding the protein of interest by PCR can becarried out by using known mRNA or cDNA encoding the protein of interestas the template according to a usual method (“PCR techniques for geneamplification—fundamental and new technologies” KYORITSU SHUPPAN, 1992,etc.).

The DNA encoding the protein of interest can also be chemicallysynthesized by the usual method, based on the nucleotide sequenceencoding the protein of interest.

AILIM of the invention(particularly preferably the human AILIM) or aportion thereof (preferably, the extracellular region) can be preparedas a recombinant protein according to a usual method with commonly usedgenetic recombination techniques, using DNA obtained by cutting DNAencoding AILIM (cDNA or intron-containing genomic DNA) based on themethod illustrated above with appropriate restriction enzymes to give aDNA fragment encoding the AILIM, and then as required, ligating theresultant DNA fragment with a linker DNA or tag, by using an appropriateDNA polymerase or the like.

The AILIM ligand (particularly preferably the human AILIM ligand) or aportion thereof (preferably, the extracellular region) can be preparedin the same manner.

A specific example is illustrated below. Namely, the DNA prepared asdescribed above is inserted into a vector, which will be described laterin detail, to yield an expression vector. Then the expression vector isused to transform a host cell as described below to obtain atransformant. The transformant is cultured and allowed to produce theprotein of interest into the culture supernatant. The protein ofinterest in the culture supernatant can easily be purified by usingcolumn chromatography and such.

There is no particular limitation on the type of expression vector forthe production of the recombinant AILIM (or its extracellular region),as far as the vector is replicated and maintained or producedautonomously in any of various hosts such as prokaryotic cells and/oreukaryotic cells. Such expression vectors include plasmid vectors andphage vectors (Cloning Vectors: A Laboratory Manual, Elsevier, New York,1985).

The expression vector can easily be prepared by ligating the DNAencoding AILIM (or its extracellular region) with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhedrosis virus.

Plasmid vectors are useful, when a DNA encoding AILIM of the invention(particularly preferably the human AILIM) or its soluble extracellularregion is intended to be expressed in a host cell and thereby expressingthe AILIM on the surface of the host cell, or alternatively the solubleextracellular region of the AILIM (particularly preferable the humanAILIM) is intended to be produced. There is no particular limitation onsuch plasmid vectors, as far as the vectors can express the geneencoding AILIM (particularly preferably the human AILIM) or its solubleextracellular region and produce the encoded protein in various hostcells such as prokaryotic cells and/or eukaryotic cells. For example,such plasmids include pMAL C2, pcDNA3.1 (−), pEF-BOS (Nucleic AcidResearch, Vol. 18, p. 5322, 1990; etc.), pME18S (“Handbook for geneticengineering,” Experimental Medicine, supplement, 1992; etc.), etc.

When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter-operator region, an initiation codon, the DNA encoding theprotein of the present invention, termination codon, terminator region,and replicon.

When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the AILIM (particularly preferablyhuman AILIM) of the present invention or its extracellular region, and atermination codon. It may also comprise the DNA encoding a signalpeptide, enhancer sequence, 5′- and 3′-untranslated region of the geneencoding the AILIM of the present invention, splicing junctions,polyadenylation site, selectable marker region, and replicon. Theexpression vector may also contain, if required, a gene for geneamplification (marker) that is usually used.

A promoter-operator region to express the AILIM (particularly preferablyhuman AILIM) of the present invention or its extracellular region inbacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD)sequence (for example, AAGG). For example, when the host is Escherichia,it preferably comprises Trp promoter, lac promoter, recA promoter, λPLpromoter, lpp promoter, tac promoter, or the like.

Examples of a promoter to express the AILIM (particularly preferablyhuman AILIM) of the present invention or its extracellular region inyeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, andso on. When the host is Bacillus, examples thereof are SL01 promoter,SP02 promoter, penP promoter and so on.

When the host is a eukaryotic cell such as a mammalian cell, examplesthereof are SV40-derived promoter, retrovirus promoter, heat shockpromoter, and so on. As a matter of course, the promoter is not limitedto the above examples. In addition, to use an enhancer is effective forexpression.

A preferable initiation codon is, for example, a methionine codon (ATG).

The commonly used termination codon (for example, TAG, TGA, TAA, and soon) is illustrated as a termination codon.

Usually used natural or synthetic terminators are used as a terminatorregion.

A replicon means a DNA capable of replicating the whole DNA sequence inhost cells, and includes a natural plasmid, an artificially modifiedplasmid (DNA fragment prepared from a natural plasmid), a syntheticplasmid, and so on. Examples of a preferable plasmids are pBR322 or itsartificial derivatives (DNA fragment obtained by treating pBR322 withappropriate restriction enzymes) for E. coli, yeast 2μ plasmid or yeastchromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145,pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, pSV2bsr, etc. for mammaliancells.

An enhancer sequence, polyadenylation site, and splicing junction thatare usually used in the art, such as those derived from SV40 can be alsoused.

A selectable marker usually used can be used according to the usualmethod. Examples thereof are resistance genes for antibiotics, such astetracycline, ampicillin, or kanamycin, and thymidine kinase gene.

Examples of a gene for gene amplification are dihydrofolate reductase(DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamatesynthase gene, adenosine deaminase gene, ornithine decarboxylase gene,hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene,etc.

The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA encoding the protein of the presentinvention, termination codon, and terminator region, to an appropriatereplicon. If desired, appropriate DNA fragments (for example, linkers,restriction sites generated with other restriction enzyme), can be usedby the usual method such as digestion with a restriction enzyme orligation using T4 DNA ligase.

Transformants of the present invention can be prepared by introducingthe expression vector mentioned above into host cells.

Host cells used in the present invention are not limited as long as theyare compatible with an expression vector mentioned above and can betransformed. Examples thereof are various cells such as natural cells orartificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insectcells.

E. coli or animal cells are preferably used. Specific examples are E.coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.), mouse-derived cells(COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc.), rat-derived cells,hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1,COS3, COS7, CV1, Velo, etc.), and human-derived cells (Hela, diploidfibroblast-derived cells, myeloma, Namalwa, etc.).

An expression vector can be introduced (transformed (transduced)) intohost cells by known method.

Transformation can be performed, for example, according to the method ofCohen et al. (Proc. Natl. Acad. Sci. USA, Vol. 69, p. 2110 (1972)),protoplast method (Mol. Gen. Genet., Vol. 168, p. 111 (1979)), orcompetent method (J. Mol. Biol., Vol. 56, p. 209 (1971)) when the hostsare bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen etal. (Proc. Natl. Acad. Sci. USA, Vol. 75, p. 1927 (1978)), or lithiummethod (J. Bacteriol., Vol. 153, p. 163 (1983)) when the host isSaccharomyces cerevisiae, the method of Graham (Virology, Vol. 52, p.456 (1973)) when the hosts are animal cells, and the method of Summerset al. (Mol. Cell. Biol., Vol. 3, pp. 2156-2165 (1983)) when the hostsare insect cells.

The extracellular region of the AILIM (particularly preferably humanAILIM) of the present invention (soluble AILIM) can be produced bycultivating transformants (in the following this term includestransductants) comprising an expression vector prepared as mentionedabove in nutrient media. AILIM ligand can be produced in the same way.

The nutrient media preferably comprise carbon source, inorganic nitrogensource, or organic nitrogen source necessary for the growth of hostcells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, etc.).

Cultivation is performed by a method known in the art. Cultivationconditions such as temperature, pH of the media, and cultivation timeare selected appropriately so that the protein of the present inventionis overproduced.

Specific media and cultivation conditions used depending on host cellsare illustrated below, but are not limited thereto.

When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi,liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used.

When the host is E. coli, examples of preferable media are LB media, M9media (Miller et al. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431 (1972)), YT media, etc. Using these media, cultivation can beperformed usually at 14 to 43° C. for about 3 to 24 hours with aerationand stirring, if necessary.

When the host is Bacillus, cultivation can be performed usually at 30 to40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

When the host is yeast, examples of media are Burkholder minimal media(Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505 (1980)). The pHof the media is preferably 5 to 8. Cultivation can be performed usuallyat 20 to 35° C. for about 14 to 144 hours with aeration and stirring, ifnecessary.

When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science, Vol. 122, p. 501(1952)), DMEM media (Virology, Vol. 8, p. 396 (1959)), RPMI1640 media(J. Am. Med. Assoc., Vol. 199, p. 519 (1967)), 199 media (Proc. Soc.Exp. Biol. Med., Vol. 73, p. 1 (1950)), HamF12 media, etc. The pH of themedia is preferably about 6 to 8. Cultivation can be performed usuallyat about 30 to 40° C. for about 15 to 72 hours with aeration andstirring, if necessary.

When the host is an insect cell, an example of media is Grace's mediacontaining fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p.8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivation canbe performed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

The extracellular region (soluble AILIM) of AILIM of the invention(particularly preferably the human AILIM) can be produced by culturingthe above-mentioned transformed cells (particularly, animal cell or E.coli) and allowing the secretion of the protein in the culturesupernatant. Namely, a culture filtrate (supernatant) is obtained by themethod such as filtration or centrifugation of the obtained culture, andthe polypeptide or polypeptide fragment of the present invention ispurified and isolated from the culture filtrate by the usual methodcommonly used in order to purify and isolate a natural or syntheticprotein.

Examples of the isolation and purification method are a method utilizingspecific affinity, such as affinity chromatography, a method utilizingsolubility, such as salting out and solvent precipitation method, amethod utilizing the difference in molecular weight, such as dialysis,ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis, a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography,a method utilizing the difference in hydrophobicity, such as reversephase high performance liquid chromatography, and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

When the protein of interest exists in the periplasm or cytoplasm ofcultured transformants, first, the fungus bodies or cells are harvestedby the usual method such as filtration or centrifugation and suspendedin appropriate buffer. After the cell wall and/or cell membrane of thecells and so on are disrupted by the method such as lysis withsonication, lysozyme, and freeze-thawing, the membrane fractioncomprising the polypeptide of the present invention is obtained by themethod such as centrifugation or filtration. The membrane fraction issolubilized with a detergent such as Triton-X100 to obtain the crudeextract. Finally, the polypeptide or the polypeptide fragment isisolated and purified from the crude extract by the usual method asillustrated above.

In the present invention, the term “insoluble carrier” means a carrierwhich is used to immobilize polypeptides on them by physical adsorptionor chemical linking. For example, the carrier can be (1) plate, testtube, tube, or the like having internal space, bead, ball, filter,membrane, or the like made of water-insoluble materials includingplastics such as polystyrene resin, polycarbonate resin, silicon resinor nylon resin, or glass, and (2) insoluble carrier used in affinitychromatography such as cellulose carrier, agarose carrier,polyacrylamide carrier, dextran carrier, polystyrene carrier,polyvnylalcohol carrier, poly amino acid carrier, porous silica carrier,etc.

The “labeling substance capable of giving detectable signal” inaccordance with the present invention includes, for example, enzyme,fluorescent material, luminescent material, biotin, avidin orradioisotope, more specifically, enzymes such as peroxidase (e.g.,horseradish peroxidase), alkaline phosphatase, β-D-galactosidase,glucoseoxidase, glucose-6-phosphate dehydrogenase, alcoholdehydrogenase, malate dehydrogenase, penicillinase, catalase,apo-glucose oxidase, urease, luciferase, acetylcholine esterase, etc.;fluorescent materials such as fluorescein isothiocyanate, phycobilinprotein, rare earth metal chelating agents, dansyl chloride, tetramethylrhodamine isothiocyanate, etc.; radioisotopes such as ³H, ¹⁴C, ¹²⁵I,¹³¹I, etc.; biotin, avidin, and luminescent material.

Among them, radioisotope or fluorescent material can give detectablesignal even when used alone. On the other hand, when used alone, enzyme,luminescent material, biotin or avidin provide no detectable signal, butwhen allowed to react with one or more substances, it can providedetectable signal. For example, when the label is an enzyme, at least asubstrate is necessary for the detection. Various types of substratescan be used depending on the type of method for measuring enzymeactivity (colorimetry, fluoroscopy, method using bioluminescence orchemical luminescence, etc.). For example, when the label is peroxidase,hydrogen peroxide can be used as a substrate. Alternatively, when thelabel is biotin, avidin or enzyme-modified avidin is commonly used butis not limited to them. As required, a variety of luminescent substancescan be utilized depending on the type of substrate to be used.

Any of the above-mentioned labels can be utilized in the presentinvention. However, preferred label is an enzyme such as peroxidase orbiotin with consideration given to sensitivity of detection or assay aswell as the convenience of manipulation.

A “method for identifying a substance capable of binding to AILIM orAILIM ligand” in accordance with the invention is constructed based onthe principle of immunoassay.

Specifically, the principles of various methods as described in“Immunoassay (3^(rd) Edition, eds., Eiji Ishikawa et al, Igakushoin,1987)” can be applied.

Examples of principles preferably used include solid-phase one-antibodymethod, liquid-phase two-antibody method, solid-phase two-antibodymethod, sandwich method, and one-pot method as described in ExaminedPublished Japanese Patent Application (JP-B) No. Hei 2-39747. Further,assay method employing antigen-antibody reaction is exemplified by EMITmethod (enzyme multiplied immunoassay technique), enzyme channelingimmunoassay, enzyme modulator mediated enzyme immunoassay (EMMIA),enzyme inhibitor immunoassay, immunoenzymometric assay, enzyme enhancedimmunoassay and proximal linkage immunoassay.

In the present invention, any of such principles of immunoassay may beselected properly in accordance with the purpose. However, withconsideration given to the convenience of procedure and/or economicadvantage, and particularly clinical versatility, the principle ofsandwich method, one-pot method, or solid-phase one-antibody method,more preferably sandwich method or one-pot method is preferablyutilized. Particularly preferred is sandwich method using multi-wellmicrotiter plate having many wells such as 96-well microplate, orone-pot method using beads on which polypeptide is immobilized and alsousing a counterpart labeled with enzyme such as peroxidase or withbiotin.

The human monoclonal antibodies of the invention capable of binding tohuman AILIM are of human origin, and therefore these antibody induces noserious immunorejection due to antigenicity to human, i.e., HAMA (Humananti-mouse antigenicity) in a host, which has been a serious therapeuticproblem (side effect) in antibody pharmaceutical preparations comprisingnon-human mammal-derived antibody such as mouse-derived antibody. Theantibody of the invention is thus of great value as an antibodypharmaceutical.

Thus, the human monoclonal antibody of the invention against AILIM(particularly human AILIM) and pharmaceutical compositions comprisingthe human monoclonal antibody do not induce host immunorejection causedby HAMA at all; and thus can be used as pharmaceutical preparationscapable of controlling a variety of biological reactions (e.g.,proliferation of cells expressing AILIM, cytokine production byAILIM-expressing cells, immune cytolysis or cell death (apoptosis) ofcells expressing AILIM, and activity of inducing antibody-dependentdamage of cells expressing AILIM) associated with the transduction ofAILIM-mediated costimulatory signal (secondary signal) to cellsexpressing AILIM; and/or can be used to treat or prevent variousdiseases associated with the transduction of the AILIM-mediated signal,controlling and inhibiting the onset and/or progress of the diseases.

Specifically, by providing pharmaceutical preparations containing thehuman anti-AILIM monoclonal antibody of the invention or a portionthereof as an active ingredient, it is possible to inhibit or treat andprevent, for example, a variety of diseases (e.g., rheumatoid arthritis,multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis,lichen planus as a chronic inflammatory skin disease, systemic lupuserythematosus, insulin dependent diabetes mellitus and psoriasis, etc.)classified into autoimmune diseases or allergic diseases (particularly,autoimmune diseases and delayed allergies by cellular immunity);arthropathies (e.g., rheumatoid arthritis (RA), osteoarthritis (OA)),inflammation (e.g., hepatitis); graft versus host reaction (GVHreaction); graft versus host disease (graft versus host disease; GVHD);immunorejection associated with transplantation (allogenic graft orheterogenous graft) of tissues (tissues such as skin, cornea and bone)or organs (liver, heart, lung, kidney, pancreas, etc.); immune responseto foreign antigen or self antigen (for example, production of antibodyagainst the antigen, cell proliferation, cytokine production, etc.); anddiseases that are potentially caused by abnormality in gut immunity(specifically, inflammatory bowel disease (particularly, Crohn's diseaseand ulcerative colitis); and alimentary allergy, etc.

The pharmaceutical compositions in accordance with the present inventionmake it possible to treat or prevent some inflammations for whichvarious steroidal drugs are used as anti-inflammatory drugs, forexample, inflammation associated with various arthritides (rheumatoidarthritis, osteoarthritis, etc.), pneumonia, hepatitis (including viralhepatitis), inflammation associated with infectious diseases,inflammatory bowel disease, enteritis, nephritis (glomerular nephritis,inflammation associated with kidney fibrosis, gastritis, vasculitis,pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis,inflammation associated with ischemia-reperfusion injury (myocaridialischemia-reperfusion injury, etc.), inflammation associated withimmunorejection after transplantation of tissues or organs, scald,various skin inflammations (psoriasis, allergic contact dermatitis,lichen planus as a chronic inflammatory skin disease), inflammationassociated with multiple organ failure, inflammation after operation ofPTCA or PTCR, and inflammation associated with atherosclerosis,autoimmune thyroiditis, etc.

In addition, with respect to the above-mentioned inhibition andtreatment of immunorejection associated with transplantation of tissuesor organs as described above, the pharmaceutical compositions inaccordance with the present invention can be used in conjunction withknown immunosuppressant used to inhibit the immunorejection intransplantation therapy thereby increasing the effect of the known drugto inhibit the graft rejection.

Further, by the use of the method of identifying substances capable ofbinding to AILIM or AILIM ligand, which is within the scope of thepresent invention, it is possible to control the signal transductionassociated with the interaction between AILIM and AILIM ligand throughthe binding to AILIM or AILIM ligand, and thereby achieving screeningand selection of pharmaceutical agents (synthetic chemical compound andantibody) having potential activity to treat the above-mentioned variousdiseases.

The present invention is illustrated in more detail below with referenceto Examples, but is not to be construed as being limited thereto.

EXAMPLE 1 Preparation of Immunogen <1-1> Preparation of Recombinant CellExpressing Human AILIM

Two types of recombinant cells (CHO cell and HPB-ALL cell)overexpressing human AILIM were prepared according to the method asdescribed in earlier applications (JP-A No. Hei 11-29599 andWO98/38216), as well as in a previous report (Int. Immunology, Vol. 12,No. 1, p. 51-55, 2000) of one of the present inventors, Tezuka.Specifically, the method is as follows:

A cDNA (GenBank Accession Number: AB023135 (cDNA); BAA82129 (amino acid)) containing the full-length ORF encoding human AILIM was inserted intoa vector pEF-neo. Then the resulting recombinant expression vector wasintroduced into Chinese hamster ovary cells (CHO cell) and cells of ahuman thymoma line, HPB-ALL, according to a commonly used method usingelectroporation (960 μF, 320V) with a Gene Pulser (BioRad). Respectivecells were cultured in RPMI1640 medium containing Geneticin (0.8 mg/ml;Gibco BRL) and 10% FCS to select drug-resistant transformed cells.

<1-2> Selection of Recombinant HPB-ALL Cells Overexpressing Human AILIM

Culture of the drug-resistant HPB-ALL cells selected in <1-1> describedabove were centrifuged to give cell pellets. A mouse anti-human AILIMmonoclonal antibody named “SA12” (mouse anti-human JTT-1 antigenmonoclonal antibody), which had been established and reported previously(JP-A 11-29599 (Example 12) and WO98/38216 (Example 12)) by the presentinventors, was added to the cell pellet (concentration: antibodysolution (10 μg/ml) diluted with EDTA-BSA/PBS was added at a ratio of100 μl/10⁵ cells). The resulting mixtures were incubated at 4° C. for 30minutes. The cells were washed twice with above-mentioned EDTA-BSA/PBS(200 ml), and then phycoerythrin-labeled streptavidin (SA-PE; 100 μl of500-fold diluted solution) was added thereto. Resulting mixtures wereincubated at 4° C. for 30 minutes. After incubation, cells were washed 3times with EDTA-BSA/PBS, and cell suspensions were prepared.

Expression levels of human AILIM of respective cells in the cellsuspensions were analyzed in a flow cytometer, FACSort(Beckton-Dichinson), to select recombinant HPB-ALL cells overexpressinghuman AILIM. Selected cells were cultured to confluence in RPMI1640medium containing 10% FCS and G418 (1 mg/ml).

<1-3> Selection of Recombinant CHO Cells Overexpressing Human AILIM

Culture of the drug-resistant CHO cells selected in <1-1> describedabove were centrifuged to give cell pellets. The above-mentioned mouseanti-human AILIM monoclonal antibody SA12, which had been labeled withFITC, was added to each cell pellet (antibody solution (100 μg/ml)diluted with EDTA-BSA/PBS). Resulting mixtures were incubated at 4° C.for 30 minutes. Cells were washed with above-mentioned EDTA-BSA/PBS, andthen cell suspensions were prepared by adding EDTA-BSA/PBS (500 μl) tothe cell pellets.

Expression levels of human AILIM of respective cells in the cellsuspensions were analyzed in a flow cytometer, FACSort(Beckton-Dichinson), to select recombinant HPB-ALL cells overexpressinghuman AILIM. Selected cells were cultured to confluence in RPMI1640medium containing 10% FCS and G418 (1 mg/ml).

<1-4> Preparation of Immunogen from HPB-ALL Cells Overexpressing HumanAILIM

The HPB-ALL cells overexpressing human AILIM, which had been obtained in<1-2> described above, were centrifuged. Recovered cell pellet waswashed 4 times with phosphate buffer (PBS; Nikken Seibutsu) and thenresuspended in a protease inhibitor-containing buffer (containing 25 mMHEPES (pH 7.4), 10 mM MgCl₂, 0.25 M Sucrose, and protease inhibitor (10U/ml Aprotinine, 2 μg/ml Pepstatin, 50 μg/ml Leupeptin, and 0.35 mg/mlPMSF)). Cell suspension was treated in a Potter-type homogenizer, andcentrifuged at a low speed (at 1,500 rpm at 4° C. for 10 minutes).Subsequently, resulting supernatant was subjected to ultracentrifugation(under 100,000 g at 4° C for 1 hour). Precipitated membrane fraction wasrecovered, and suspended in phosphate buffer (concentration of themembrane fraction was adjusted so that 1 ml PBS contains membranefraction derived from 1×10⁷ cells). The suspension was stored at −80° C.The suspension containing cell membrane fraction was used as the antigen(immunogen) to prepare human antibody of the present invention, whichwill be described later.

<1-5> Preparation of Immunogen from CHO Cells Overexpressing Human AILIM

The CHO cells overexpressing human AILIM, which had been obtained in<1-3> described above, were dispersed with a scraper and werecentrifuged. Recovered cell pellet was washed 4 times with phosphatebuffer (PBS; Nikken Seibutsu) and then resuspended in a proteaseinhibitor-containing buffer (containing 25 mM HEPES (pH 7.4), 10 mMMgCl₂, 0.25 M Sucrose, and protease inhibitor (10 U/ml Aprotinine, 2μg/ml Pepstatin, 50 μg/ml Leupeptin, and 0.35 mg/ml PMSF)). The cellsuspension was treated in a Potter-type homogenizer, and centrifuged ata low speed (at 1,500 rpm at 4° C. for 10 minutes). Subsequently,resulting supernatant was subjected to ultracentrifugation (under100,000 g at 4° C. for 1 hour). Precipitated membrane fraction wasrecovered, and suspended in phosphate buffer (concentration of themembrane fraction was adjusted so that 1 ml PBS contains membranefraction derived from 1×10⁷ cells). The suspension was stored at −80° C.The suspension containing cell membrane fraction was used as the antigen(immunogen) to prepare human antibody of the present invention, whichwill be described later.

EXAMPLE 2 Preparation of Hybridoma Producing Human Anti-Human AILIMMonoclonal Antibody

In the present Example, preparation of monoclonal antibody was carriedout according to a typical method as described in “Experimental Medicine(supplement), Handbook for Cell Technology” (eds., T. Kuroki et al.,Yodosha, pp. 66-74, 1992) and “Experimental Manual for MonoclonalAntibody” (T. Ando et al., Kodansha, 1991).

Cell membrane fraction prepared from recombinant cells overexpressinghuman AILIM provided in Example 1 was used as the immunogen of humanAILIM.

Animals subjected to immunization were human antibody-producingtransgenic mice created by above-described method (Nature Genetics, Vol.7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; PublishedJapanese Translation of International Application No. Hei 4-504365;Published Japanese Translation of International Application No. Hei7-509137; Nikkei Science, June, pp. 40-50, 1995; etc.).

Multi-well microplates were used for cell culture.

<2-1> Immunization and Preparation of Hybridoma

Either of the immunogens (100 μl/mouse/administration) prepared in <1-4>(derived from HPB-ALL) and in <1-5> (derived from CHO) described abovewas given to the above-mentioned human antibody-producing transgenicmouse. The immunogen was injected together with Freund's completeadjuvant (ICN/CAPPEL) in the footpad as primary immunization (day 0).

After primary immunization, either of the two immunogens wasadditionally injected to the footpad at 1-week interval as secondaryand/or tertiary immunization. In the same manner, injection was furthercarried out for final immunization two days before preparation oflymphocytes, which is described below.

Two days after final immunization, lymphocytes were prepared from(subinguinal and subgenual) lymph nodes and spleens of respectivetransgenic mice subjected to immunization. The lymphocytes and mousemyeloma cells P3/X63-AG8.653 (ATCC No. CRL-1580) were mixed at a ratioof 5:1, and polyethylene glycol 1500 (Boehringer Mannheim) was addedthereto as a fusion agent. Then, the mixture was diluted with 10 volumesof serum-free basal medium EX-CELL301 (JRH Bioscience). Subsequently,the mixed cells were washed with the basal medium and then suspended inHAT medium (1 L of basal medium contained 13.61 mg of hypoxanthine, 176μg of aminopterin, and 3.88 mg of thymidine). The cells were plated on96-well microplates and cultured for 10-14 days to complete cell fusion.The cell fusion treatment yielded many hybridomas.

<2-2> Screening of Human Monoclonal Antibody-Producing Hybridoma

A number of hybridomas prepared in <2-1> described above were screenedwith cell ELISA as described below to select hybridomas producing humanmonoclonal antibody against human AILIM.

Respective recombinant HPB-ALL cells and recombinant CHO cellsoverexpressing human AILIM, which are described above, were plated ineach well of ELISA 96-well microplates (1×10⁵ cell/well). Incubation wascarried out at 37° C. for 2 days.

Subsequently, supernatant of each well was discarded, samples ofsupernatant of each hybridoma culture was added thereto (50 μl/well),and the mixture was incubated for 1 hour. After the reaction wascompleted, the mixed sample solution was discarded and each well waswashed 3 times with PBS containing 1% BSA (Sigma).

Subsequently, peroxidase-conjugated goat anti-human immunoglobulin (Fc)antibody (50 μl of the 2000-fold dilute per well; Ameircan Corex; 1%BSA/PBS) was added to each well in order to detect the heavy chain ofhuman immunoglobulin (human monoclonal antibody) in the hybridomasupernatant. The mixture was incubated at room temperature for 1 hour.

On the other hand, peroxidase-conjugated goat anti-human immunoglobulinκ chain antibody (50 μl of the 2000-fold dilute per well) was added toeach well in order to detect the light chain of human immunoglobulin(human monoclonal antibody) in the hybridoma supernatant. The mixturewas incubated at room temperature for 15 minutes.

The anti-human IgFc antibody or anti-human Igκ antibody was removed fromeach well of the microplates, and then the plates were washed 3 timeswith PBS containing 1% BSA. Tetramethylbenzidine(3,3′,5,5′,-tetramethylbenzidine (TMB), 100 μl/well, BIO-RAD) was addedto each well, and the resulting mixture was incubated at roomtemperature for 15 minutes.

Subsequently, 1N H₂SO₄ was added to each well (50 μl/well) to quench thereaction. The reaction was monitored for absorbance at a wavelength of450 nm by a microplate reader (Model 3550 Microplate Reader, BIO-RAD).

Control ELISA experiment was performed in the same manner as describedabove by using the following items:

(1) Wild-type HPB-ALL cells, instead of human AILIM expressingrecombinant HPB-ALL cells;

(2) Wild-type CHO cells, instead of human AILIM expressing recombinantCHO cells;

(3) Mouse monoclonal antibodies against human AILIM (SA12 or SG430; JP-A11-29599 (Example 12) and WO98/38216 (Example 12)) instead of hybridomasupernatant;

(4) Human monoclonal antibody against KLH (keyhole limpet hemocyanin,PIERCE) instead of hybridoma supernatant.

The human anti-KLH monoclonal antibody was prepared according to thesame manner as described above in <2-1>, by immunizing above-mentionedhuman antibody-producing transgenic mice with KLH (keyhole limpethemocyanin, PIERCE).

Many hybridomas producing human monoclonal antibody capable of bindingto human AILIM were selected by said screening.

<2-3> Primary Cloning of Hybridoma

Many types of hybridoma monoclones were established from the varioushybridomas (parental cell lines), which had been selected in <2-2>described above, producing human monoclonal antibody against human AILIMby the following assay procedure.

Respective hybridomas selected in <2-2> described above were plated in24-well microplates. Cell count of hybridoma in each well was determinedby pipetting. Subsequently, 10% fetal calf serum (FCS; Trace BiosciencePTY), 1% penicillin/streptomycin (Sigma), 1% HT Supplement (Gibco BRL)and 2.5% T-STIM Culture Supplement (Collaborative Biomedical Products)were added to EX-CELL301 medium (JRH Bioscience) containing 4.0 mML-glutamine and lipid. The resulting modified medium was used to dilutehybridomas to 1×10⁴ cells/ml and the cells were suspended in each well.

Cell suspension (300 μl or 600 μl) of each well was combined and mixedwell with the modified medium (150 ml or 300 ml), and then a 200-μlaliquot of the cell suspension was added to each well of multiple96-well microplates such that each well contained 4 cells of thehybridoma. The above-mentioned modified medium (50 ml or 100 ml) wasfreshly added to the remaining cell suspension, which was mixed well,and then the resulting cell suspension was added to each well of otherfreshly prepared multiple 96-well microplates such that each wellcontained 2 cells of the hybridoma.

Cultivation was continued for 1 to 2 weeks. After cultivation, singlecolonies derived from a single hybridoma were found in many wells.

With the cell ELISA as described above in <2-2>, it was verified thathuman monoclonal antibody against human AILIM was produced in theculture supernatant in each well containing the colony.

<2-4> Secondary Cloning of Hybridoma

Subcloning (secondary cloning) of each clone from the various hybridomaclones obtained in <2-3> described above was performed according to thesame method as described above in <2-3>.

Cell density of each well in a 96-well microplate was adjusted to 1cell/well in the present experiment.

The screening yielded many hybridoma monoclones producing humanmonoclonal antibody against human AILIM. Part of the clones includedwere as below:

(Clone Name)

AIF34 (JMab124), AIF182 (JMab-126), AIF348 (JMab-127),

AIF620 (JMab-128), AIF1052 (JMab-135), AIH5D3 (JMab-136),

AIH386 (JMab-137), AII289 (JMab-138), AII394 (JMab-139),

AII488 (JMab-140), AIJ40 (JMab-141),

The names shown above are used through the present application, namelyin all Examples described below including the present Example, and inFigures and Tables containing the assay result obtained in this Example.

EXAMPLE 3 Analysis of Properties of Monoclonal Antibody <3-1> Analysesof the Heavy Chain and Light Chain

By using ELISA and flow cytometry described below, it was verified thatthe monoclonal antibody against human AILIM produced by each hybridomaclone, which had been cloned in <2-4> described above, was indeed ahuman monoclonal antibody.

The recombinant HPB-ALL cells overexpressing human AILIM, which had beenprepared in Example 1, were plated in each v-shaped well of microplate(3×10⁴ cell/well). Cells were cultured at 37° C. in RPMI1640 mediumcontaining 10% FCS.

After the culture was completed, the plate was centrifuged (at 1,800 rpmfor 2 minutes) to precipitate the cells, and then the resultingsupernatant was discarded. Subsequently, supernatant sample (50 ml/well)from the culture of each hybridoma cloned in <2-4> described above, ormouse anti-human AILIM monoclonal antibody SA12 (2 μg/50 μl) oralternatively human anti-KLH monoclonal antibody (50 μl/well) as acontrol antibody was added to each well. The mixture was reacted for 30minutes in a refrigerator. After reaction, the sample solution wasdiscarded and each well was washed with phosphate buffer (0.5% BSA-PBScontaining 5 mM EDTA).

Subsequently, any one of the secondary antibodies below was added toeach well (diluted 1000 times with the above phosphate buffer and addedat a quantity of 50 μl/well) in order to suspend the cells. Thesuspension was reacted for 30 minutes in a refrigerator.

(Secondary Antibody)

Biotin-labeled anti-human IgG antibody (Zymed);

Biotin-labeled anti-human IgG antibody (Protos);

Biotin-labeled anti-human IgFc antibody (EY Laboratories); or

Biotin-labeled anti-human Igκ antibody (Vector).

After reaction, the secondary antibody was discarded and each well ofthe plate was washed with the above-mentioned phosphate buffer.Subsequently, phycoerythrin-labeled streptavidin (Streptavidin-PE;Pharmingen; diluted 500 time with the above-mentioned phosphate bufferand added at a quantity of 50 μl/well) was added to each well. Themixture was reacted for 30 minutes in a refrigerator. After reaction,each well was washed with the above-mentioned phosphate buffer. Then,the above-mentioned phosphate buffer was added to each well (200μl/well) in order to suspend the cells.

Analysis was performed to determine the reactivity of the anti-humanAILIM monoclonal antibody in the culture supernatant of each hybridomaclone to the HPB-ALL cells overexpressing human AILIM in each well.

Control assay was performed in the same manner as described above byusing following items:

(1) Wild-type HPB-ALL cells, instead of human AILIM expressingrecombinant HPB-ALL cells;

(2) Human monoclonal antibody against KLH (keyhole limpet hemocyanin,PIERCE) instead of hybridoma supernatant.

The human anti-KLH monoclonal antibody was prepared according to thesame manner as described above in <2-1>, by immunizing theabove-mentioned human antibody-producing transgenic mice with KLH(keyhole limpet hemocyanin, PIERCE).

Based on the result, all of the hybridoma clones described above in<2-4> were verified to be human monoclonal antibodies consisting ofhuman-derived heavy chain and human-derived κ light chain.

An example of the result is illustrated in FIG. 1, which involves assayresult for hybridoma clones AIH5D3 (JMab-136), AII289 (JMab-138), andAII394 (JMab-139).

<3-2> Isotyping of Human Monoclonal Antibody

The isotype was determined for each of the human anti-human AILIMmonoclonal antibodies produced by the hybridomas that had been cloned in<2-4> and analyzed in <3-1> described above. Determination was carriedout using a Human Monoclonal Antibody Isotyping Kit (American Qualex)according to the experimental protocol attached to the kit.

All the human anti-human AILIM monoclonal antibodies were determined tobe IgG2/κ.

EXAMPLE 4 Preparation of Human Monoclonal Antibody Against Human AILIM(Human Anti-Human AILIM Monoclonal Antibody) on Large Scale and itsPurification <4-1> Method 1

Cells of each hybridoma clone producing human anti-human AILIMmonoclonal antibody, which had been prepared in <2-4> described above,were added to a tissue culture flask (50 ml, FALCON), and cultured inASF104 medium (Ajinomoto) containing 10% Ultra Low Bovine IgG FBS(GIBCO-BRL) to be confluence under an atmosphere of 5% CO₂ at 37° C.

Subsequently, the whole culture liquid was transferred into a new tissueculture flask (750 ml, FALCON), and the cells were cultured in ASF104medium (Ajinomoto) containing 10% Ultra Low Bovine IgG FBS (GIBCO-BRL)to be confluent under an atmosphere of 5% CO₂ at 37° C.

10 to 20 days after the culture, the culture supernatant of eachhybridoma was recovered and transferred into a 50-ml polypropyleneconical tube (FALCON). The tube was centrifuged under 500×g for 5minutes.

Subsequently, resulting centrifugal supernatant was filtered through aSterilization Filter Unit (NALGEN), and the filtrate was recovered.

The filtrate was loaded onto a HiTrap Protein G column (HiTrap affinitycolumn Protein G; Amersham Pharmacia) pre-equilibrated with phosphatebuffer (30 ml) at a flow rate of 3 ml/min.

Subsequently, the column was washed with phosphate buffer (20 ml), andthen the antibody of interest was eluted by loading 100 mM citratebuffer (pH 2.0) onto the column at a low rate of about 1 ml/min.Subsequently, the eluted solution was neutralized with a solution (pH9.0) of 750 mM Tris-HCl, and then filtered through a filter (Millipore)to remove white precipitate. The resulting filtrate was dialyzed againstphosphate buffer (overnight) and filtered through a filter (Millipore).Thus purified anti-AILIM human monoclonal antibody was obtained fromeach hybridoma line.

Protein concentration was determined from the absorbance at A₂₈₀measured by using a photospectrometer (1A₂₈₀=1.41 mg/ml).

<4-2> Method 2

Cells of each hybridoma clone, which had been prepared in <2-4>described above, were conditioned in ASF104 medium (Ajinomoto)containing 10% Ultra Low Bovine IgG FBS (GIBCO-BRL) (1-2×10⁶ Cells/mleach), and were plated and cultured in Integra Cell Line 1000 (INTEGRACL1000, INTEGRA BIOSCIENCE). 7 to 10 days after cultivation, when thecell density reached 1×10⁸ cells/ml, the supernatant of each hybridomaculture was recovered.

Each culture supernatant was loaded onto a HiTrap Protein G column(HiTrap affinity column Protein G; Amersham Pharmacia) pre-equilibratedwith phosphate buffer (30 ml) at a flow rate of 3 ml/min.

Subsequently, the column was washed with phosphate buffer (20 ml), andthen 100 mM citrate buffer (pH 2.0) was loaded onto the column at a flowrate of about 1 ml/min to elute the antibody. Then, a solution (pH 9.0)of 750 mM Tris-HCl was added to neutralize the eluted solution, and theresulting solution was filtered through a filter (Millipore) to removewhite precipitate. The resulting filtrate was dialyzed against phosphatebuffer (overnight) and then filtered through a filter (Millipore). Thuspurified anti-AILIM human monoclonal antibody was obtained from eachhybridoma line.

EXAMPLE 5 Reactivity of Human Anti-Human AILIM Monoclonal Antibody toHuman AILIM, and Cross-Reactivity of that to Mouse AILIM and Rat AILIM

Purified various human anti-human AILIM monoclonal antibodies above wereanalyzed for their reactivity to human AILIM as well as cross-reactivityto mouse AILIM and rat AILIM by utilizing cell ELISA method.

<5-1> Establishment of ELISA System to Determine IgG AntibodyConcentration and Preparation of Calibration Curves

Because all the above-mentioned purified human anti-human AILIMmonoclonal antibodies were IgG (IgG2) antibody, ELISA system wasestablished to determine the concentration of IgG antibody.

Goat anti-human IgG (Fc) antibody (1.2 μg/ml in PBS; 100 μl/well;Organon Teknika) was added to each well of a 96-well ELISA microplate(Nunc). The plate was incubated at room temperature for 2 hours toadsorb the anti-IgG (Fc) antibody on the microplate. Subsequently, thesupernatant was discarded, and the plate was washed 3 times withphosphate buffer (PBS) containing 0.05% Tween20. A blocking reagent (PBScontaining 0.5% bovine serum albumin (BSA) and 0.1% Tween20) was addedto each well (200 μl/well) and the plate was incubated at roomtemperature for 2 hours to block the anti-IgG (Fc) antibody-free siteson the plate. Then, the blocking reagent was discarded, and each wellwas washed twice with PBS.

Human-derived IgG2 antibody (50 μl/well; The Binding Site), which wasused as a standard antibody, was added at various concentrations (0 to100 ng/ml) to respective wells of the plate and the plate was incubatedat room temperature for 2 hours. Surplus solutions of standard antibodywere removed and each well was washed 3 times with phosphate buffercontaining 0.05% Tween20.

Subsequently, peroxidase-conjugated goat anti-human IgG/κ antibody wasadded to each well (4,000 times diluted, 100 μl/well, Protos), and theplate was incubated at room temperature for 1 hour.

The supernatant was discarded and the microplate was washed 3 times withphosphate buffer containing 0.05% Tween20. A buffer containing substrate(composition: ortho-phenylenediamine (O-phenylenediamine, OPD; 20mg)/citrate-phosphate buffer (pH 5.0, 50 ml)/aqueous solution of 30%hydrogen peroxide (15 μl)) was added to each well (100 μl/well) and theplate was incubated at room temperature for about 7 minutes.

Subsequently, 2 M sulfuric acid was added to each well (50 μl/well) tostop the reaction. Calibration curves were made (FIG. 2) based on thevalues of absorbance measured at a wavelength of 490 nm by using amicroplate reader.

Control assays were performed with culture medium alone or BSA solutionalone as a test substance in the same manner as described above.

<5-2> Analyses for the Reactivity of Various Purified Human Anti-HumanAILIM Monoclonal Antibodies to Human AILIM as Well as forCross-Reactivity of that to Mouse AILIM and Rat AILIM

<5-2-1> Preparation of Reagents

Reagents to be used in this cell ELISA were prepared as follows:

<5-2-1 -1> Preparation of Recombinant CHO Cell Overexpressing MouseAILIM

Recombinant CHO cells overexpressing mouse AILIM were prepared andobtained in the same manner as described above in <1-1> and <1-3>.

cDNA (GenBank Accession Number: AB023132 (cDNA); BAA82126 (amino acid))containing the full-length ORF of mouse AILIM was inserted into a vectorpEF-neo, and then the resulting recombinant expression vector wasintroduced into Chinese hamster ovary cells (CHO cell) by a commonlyused method for electroporation (960 μF, 320 V) using a Gene Pulser(BioRad). The cells were cultured in RPMI1640 medium containingGeneticin (0.8 mg/ml; Gibco BRL) and 10% FCS to select drug-resistanttransformed cells, thereby obtaining mouse AILIM-overexpressingrecombinant CHO cells.

<5-2-1-2> Preparation of Recombinant CHO Cell Overexpressing Rat AILIM

Recombinant CHO cells overexpressing rat AILIM were prepared andobtained in the same manner as described above in <1-1> and <1-3>.

A cDNA (GenBank Accession Number: AB023134 (cDNA); BAA82128 (aminoacid)) containing the full-length ORF of rat AILIM was inserted into avector pEF-neo, and then the resulting recombinant expression vector wasintroduced into Chinese hamster ovary cells (CHO cell) by a commonlyused method for electroporation (960 μF, 320 V) using a Gene Pulser(BioRad). The cells were cultured in RPMI1640 medium containingGeneticin (0.8 mg/ml; Gibco BRL) and 10% FCS to select drug-resistanttransformed cells, thereby obtaining rat AILIM-overexpressingrecombinant CHO cells.

<5-2-1-3> Preparation of Monoclonal Antibody Against Mouse AILIM

The recombinant CHO cells overexpressing mouse AILIM, which had beenprepared in <5-2-1-1> described above were homogenized, and subjected toultracentrifugation (100,000×g). Resulting pellet containing cellmembrane fraction was recovered and then suspended in PBS. Resultingcell membrane fraction was injected together with Freund's completeadjuvant to Wistar rats in the footpad for primary immunization (day 0).The antigen of cell membrane fraction was further given to the rats intothe footpad on the 7^(th) day, 14^(th) day and 28^(th) day after theprimary immunization. The lymph node cells were collected from them 2days after the final immunization.

The lymph node cells and mouse myeloma cell PAI (JCR No.B0113; Res.Disclosure, Vol. 217, p. 1.55, 1982) were combined at a ratio of 5:1.The cells were fused to each other by using polyethylene glycol 4000(Boehringer Mannheim) as a fusion agent to prepare monoclonalantibody-producing hybridomas. Selection of hybridomas was achieved byculturing them in ASF104 medium (Ajinomoto) containing HAT, 10% fetalcalf serum and aminopterin.

Reactivity of rat monoclonal antibody in the culture supernatant of eachhybridoma to mouse AILIM was determined by reacting the culturesupernatant with the above-mentioned CHO cells expressing mouse AILIMand then measuring the fluorescence intensity of cells stained withFITC-labeled anti-rat IgG (Cappel) in a EPICS-ELITE flow cytometer. Thescreening yielded multiple hybridomas producing monoclonal antibodyhaving reactivity against mouse AILIM.

Among them, a hybridoma line was named “B10.5.” Cells of this hybridomawere intraperitoneally injected (10⁶ to 10⁷ cells/0.5 ml/mouse) to ICRnu/nu mice (female, 7 to 8-weeks old). 10 to 20 days after theinjection, the ascites was collected from each mouse by laparotomy underanesthesia according to a commonly used method. The rat anti-mouse AILIMmonoclonal antibody B10.5 (IgG1) was prepared from the ascites on alarge scale.

<5-2-2> The Reactivity of the Antibody to Human, Mouse and Rat AILIM

Concentrations of human anti-human AILIM monoclonal antibody and controlantibody to be used in the ELISA described below were determined basedon the ELISA and calibration curves in <5-1> described above.

Each cells (7×10³ cells/well) of the recombinant CHO cell overexpressinghuman AILIM prepared in Example 1, the recombinant CHO celloverexpressing mouse AILIM prepared in <5-2-1-1> described above, andthe recombinant CHO cell overexpressing rat AILIM prepared in <5-2-1-2>described above were plated in wells of 96-well ELISA microplates andcultured to be confluent at 37° C.

Subsequently, the supernatant was discarded, and then any one of thepurified various human anti-human AILIM monoclonal antibodies or controlantibody prepared above were added to each well (antibodyconcentrations: antibody of 200 μg/ml was diluted, with PBS containing1% BSA, 3 times, 3² times, 3³ times, 3⁴ times, 3⁵ times, 3⁶ times, 3⁷times, 3⁸ times, 3⁹ times, 3¹⁰ times, 3¹¹ times, and 3¹² times) in aquantity of 50 μl/well, and the plates were reacted at room temperaturefor 2 hours. The solutions of the monoclonal antibodies were discarded,and each well was washed 3 times with phosphate buffer containing 1% BSA(Sigma).

Subsequently, horseradish peroxidase-conjugated anti-human IgG (Fc)antibody was added to each well (diluted 1,000 times, 50 μl/well;American Qualex), and the plates were incubated at room temperature for1 hour.

Surplus solution of the labeled antibody was discarded and themicroplates were washed 3 times with phosphate buffer containing 1% BSA.Buffer containing substrate (composition: ortho-phenylenediamine(O-phenylenediamine, OPD; 20 mg)/citrate-phosphate buffer (pH 5.0, 50ml)/aqueous solution of 30% hydrogen peroxide (15 μl)) was added to eachwell (100 μl/well) and the plates were incubated at room temperature forabout 7 minutes.

Subsequently, 2 M sulfuric acid was added to each well (50 μl/well) tostop the reaction. Absorbance was measured at a wavelength of 490 nm byusing a microplate reader (Bio-Rad).

Control ELISA assay was performed with the following control antibodiesto evaluate the above-mentioned antibodies in the same manner asdescribed above:

(1) Mouse monoclonal antibody SA12 or SG430 against human AILIM (JP-A11-29599 (Example 12) and WO98/38216 (Example 12));

(2) Rat monoclonal antibody B10.5 against mouse AILIM (<5-2-1-3>described above);

(3) Mouse monoclonal antibody JTT2 against rat AILIM (monoclonalantibody produced by a hybridoma, which has been depositedinternationally on Oct. 11, 1996, under the international accessionnumber FERM BP-5708 in The National Institute of Bioscience andHuman-Technology, The Agency of Industrial Science and Technology, TheMinistry of International Trade and Industry)), which is aninternational depositary authority under the Budapest Treaty; JP-A11-29599 (Examples 1 and 2) and WO98/38216 (Examples 1 and 2)).

(4) The above-prepared human monoclonal antibody against KLH (keyholelimpet hemocyanin, PIERCE) instead of hybridoma supernatant.

Control ELISA experiment was performed in the same manner as describedabove using wild-type CHO cell shown below, instead of AILIM-expressingrecombinant CHO cell.

The result is shown in FIGS. 3 to 14.

Based on the result obtained, 50%-effective concentration (ED50:ng/ml)was calculated as an index for the reactivity of each human anti-humanAILIM monoclonal antibody to human AILIM (recombinant CHO celloverexpressing human AILIM), mouse AILIM (recombinant CHO cellsoverexpressing mouse AILIM), or rat AILIM (recombinant CHO celloverexpressing rat AILIM). The results obtained by the calculation areshown below.

(A) ED50 for CHO over-expressing human AILIM

-   -   AIF 34 (JMab-124): 5.3 ng/ml    -   AIF182 (JMab-126): 3.6 ng/ml    -   AIF348 (JMab-127): 9.1 ng/ml    -   AIF620 (JMab-128): 10.1 ng/ml    -   AIF1052 (JMab-135): 2.0 ng/ml    -   AIH5D3 (JMab-136): 7.5 ng/ml    -   AIH386 (JMab-137): 9.6 ng/ml    -   AII289 (JMab-138): 10.5 ng/ml    -   AII394 (JMab-139): 10.6 ng/ml    -   AII488 (JMab-140): 11.0 ng/ml    -   AIJ 40 (JMab-141): 3.7 ng/ml    -   SA 12: 1.8 ng/ml    -   SG430: 1.2 ng/ml

(B) ED50 for CHO overexpressing mouse AILIM

-   -   AIF 34 (JMab-124): 42 ng/ml    -   AIF348 (JMab-127): 81 ng/ml    -   AIF620 (JMab-128): 100 ng/ml    -   AII289 (JMab-138): 53 ng/ml    -   AII394 (JMab-139): 60 ng/ml    -   AII488 (JMab-140): 70 ng/ml

(C) ED50 for CHO overexpressing rat AILIM

-   -   AIF 34 (JMab-124): 45 ng/ml    -   AIF348 (JMab-127): 62 ng/ml    -   AIF620 (JMab-128): 97 ng/ml    -   AII289 (JMab-138): 57 ng/ml    -   AII394 (JMab-139): 90 ng/ml    -   AII488 (JMab-140): 90 ng/ml

The result showed that the human anti-human AILIM monoclonal antibodiesof the present invention exhibited significantly high specificities tohuman AILIM.

Further, it has been revealed that 6 types of human anti-human AILIMmonoclonal antibodies (shown above in (B) and (C)) are reactive to bothmouse AILIM and rat AILIM (binding capability, cross-reactivity).

EXAMPLE 6 Determination of Affinity and Neutralizing Activity of HumanAnti-Human AILIM Monoclonal Antibody Against the Antigen (Human AILIM)

Association rate constant (ka), dissociation rate constant (kd) anddissociation constant (Kd) with respect to the reaction between each ofthe purified various human anti-human AILIM monoclonal antibodiesprepared above and human AILIM were determined using a commerciallyavailable kit Biacore X (Amersham Pharmacia).

<6-1> Preparation of Antigen to be Immobilized on Sensor Chip

Antigen to be immobilized on sensor chip in the kit was prepared as arecombinant chimeric antigen (hereinafter referred to as “humanAILIM-IgFc”) consisting of the extracellular region of human AILIM andthe constant region (Fc) of human IgG1.

Human AILIM-IgFc was prepared by further purifying the antigen obtainedaccording to the method as described in earlier applications (JP-A11-29599 (Example 16 (2)) and WO98/38216 (Example 16 (2)) by one of thepresent inventors, Tezuka.

The culture supernatant of recombinant cells producing the humanAILIM-IgFc was loaded onto a HiTrap Protein G column (HiTrap affinitycolumn Protein G; Amersham-Pharmacia) pre-equilibrated with phosphatebuffer (30 ml) at a flow rate of 3 ml/min to adsorb the human AILIM-IgFcin the culture supernatant on the column.

Subsequently, the column was washed with phosphate buffer (20 ml), andthen 100 mM citrate buffer (pH 2.0) was loaded onto the column at a flowrate of about 1 ml/min to elute the human AILIM-IgFc. Subsequently, theeluted solution was neutralized with a solution (pH 9.0) of 750 mMTris-HCl, and then dialyzed against phosphate buffer (overnight). Then,the solution dialyzed was filtered through a filter (Millipore). Thuspurified anti-human AILIM-IgFc was obtained.

Protein concentration was determined from the absorbance at A₂₈₀measured by using a photospectrometer (1A₂₈₀=1 mg/ml). The concentrationof human AILIM-IgFc was determined to be 0.28 mg/ml.

Purified chimeric protein consisting of the extracellular region of ratAILIM and the constant region (Fc) of human IgG1 (rat AILIM-IgFc; JP-A11-29599 (Example 16 (2)) and WO98/38216 (Example 16 (2)) was alsoprepared according to the same manner as described above. Theconcentration of the rat AILIM-IgFc obtained was determined to be 0.45mg/ml.

<6-2> Determination of Affinity and Neutralizing Activity

Experimental procedures except for immobilization of antigen (humanAILIM-IgFc) on the sensor chip, which is described below, were based onthe instruction manual and experimental protocol attached to thecommercially available assay kit Biacore X (Amersham-Pharmacia).

HBS buffer (containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and 0.005%detergent P20, (pH 7.0)) was allowed to flow through a Flow Cell-1attached to the kit at a flow rate of 5 μl/min. Subsequently, a solution(15 μl) containing 0.005 M NHS (N-hydroxysuccinimide) and 0.2 M EDC(N-ethyl-N′-(dimethylaminopropyl)carbodiimide) was added to activatecarboxyl groups of CM coated on the surface of the sensor chip.

Subsequently, 23 μl of human AILIM-IgFc solution (10 μg/ml; dissolved in10 mM sodium acetate buffer (pH 5.0)) was added to the to immobilize thehuman AILIM-IgFc on the sensor chip. Subsequently, unreacted activatedcarboxyl groups were blocked by adding 35 ml of 1 M ethanol aminehydrochloride. The amounts of human AILIM-IgFc immobilized by theimmobilization treatment performed twice was 2,444RU (resonance unit)and 2,213RU, respectively. The unit, RU, corresponds to the mass perunit area; 1RU=1 pg/mm².

Flow Cell-2, which is a reference flow cell, was subjected to thecapping treatment in the absence of human AILIM-IgFc in the same manneras described above.

Phosphate buffer was allowed to flow through the flow cell (sensor chip)at a flow rate of 20 μl/min, and each of purified human anti-human AILIMmonoclonal antibodies, which had been prepared in the Example above, wasadded thereto (10 to 50 μg/ml, 60 μl).

Standard condition for the measurement was: association phase for 3minutes and dissociation phase for 10 minutes. Respective amounts ofantibody bound to and released from the antigen were monitored over timeto obtain a sensorgram. Dissociation of antibody from the antigen wasachieved by running PBS through the sensor chip at a flow rate of 20μl/min.

Based on the resulting sensorgram data, association rate constant (ka),dissociation rate constant (kd) and dissociation constant (Kd; Kd=kd/ka)were computed by using the analytical software (BIAevaluation 3.0)attached to the kit.

The affinity and the neutralizing activity of mouse monoclonalantibodies SA12 and SG430 to the human AILIM prepared in the Exampledescribed above were also analyzed in the same manner as describedabove.

Respective values obtained are shown below.

-   <clone name> <ka (1/M.Sec)> <kd [1/Sec]> <Kd (M)>-   AIF34 (JMab-124) 1.6×10⁴ 1.0×10⁻⁴ 6.3×10⁻⁹-   AIF182 (JMab-126) 3.2×10⁴ 2.8×10⁻⁵ 8.8×10⁻¹⁰-   AIF348 (JMab-127) 1.9×10⁴ 6.4×10⁻⁵ 3.4×10⁻⁹-   AIF620 (JMab-128) 1.1×10⁴ 1.1×10⁻⁴ 1.0×10⁻⁸-   AIF1052 (JMab-135) 1.6×10⁴ 6.3×10⁻⁵ 3.9×10⁻⁹-   AIH5D3 (JMab-136) 2.8×10⁴ 4.9×10⁻⁶ 1.8×10¹⁰-   AIH386 (JMab-137) 1.2×10⁵ 3.1×10⁻⁴ 2.6×10⁻⁹-   AII289 (JMab-138) 3.7×10⁴ 4.2×10⁻⁵ 1.1×10⁻⁹-   AII394 (JMab-139) 3.1×10⁴ 2.4×10⁻⁵ 7.7×10⁻¹⁰-   AII488 (JMab-140) 2.3×10⁴ 3.5×10⁻⁵ 1.5×10⁻⁹-   AIJ40 (JMab-141) 1.9×10⁴ 1.9×10⁻⁵ 1.0×10⁻⁹-   SA 12 7.8×103 7.9×10⁻⁵ 1.0×10⁻⁸-   SG430 2.2×104 1.5×10⁻⁴ 6.8×10⁻⁹

The result shows that all of the human anti-human AILIM monoclonalantibodies and anti-human AILIM mouse monoclonal antibodies exhibitmarkedly high binding affinity and neutralizing activity to human AILIM.

EXAMPLE 7 Activity of Human Anti-Human AILIM Monoclonal Antibody toTransduce Costimulatory Signal in Human T Cell

It was analyzed whether or not the human anti-human AILIM monoclonalantibodies in accordance with the present invention had the capabilityof controlling (enhancing and/or inhibiting) human T cell responses(production of cytokines such as IFN-γ and IL-4, cell proliferation,etc.), in other words, whether or not the antibodies exhibitedregulatory activity on cellular transduction of AILIM-mediatedcostimulatory signal. Analysis was performed based on the amount ofcytokines (IFN-γ and IL-4) produced in human T cells as well as thedegree of human T cell proliferation as an index.

<7-1> Dilution of Antibody

Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted withphosphate buffer (PBS) to final concentration of 8 μg/ml.

Each of the various human anti-human AILIM monoclonal antibodiesprepared above was diluted with PBS to a final concentration of 40μg/ml. The antibody solutions were further diluted with PBS to preparevarious concentrations of antibodies (40 μg/ml-0.0049 μg/ml).

<7-2> Coating of Microplate with Antibody

Each well of 96-well microplates was coated with (1) anti-human CD3monoclonal antibody OKT3 (8 μg/ml; 25 μl to each well) and any one ofthe various human anti-human AILIM monoclonal antibodies (40μg/ml-0.0049 μg/ml; 25 μl to each well), or (2) anti-human CD3monoclonal antibody OKT3 (8 μg/ml; 25 μl to each well) alone. The plateswere incubated at 37° C. for 2 hours. Subsequently, the antibodysolutions were discarded, and each well was washed 3 times with PBS.After the wash, RPMI1640 medium containing 10% FCS was added to eachwell (100 μl/well), and the plates were incubated at 37° C. for 1 hour.Thus, respective wells of the plates were coated with the antibodiesmentioned above in (1) or (2).

Control experiments were carried out in the same manner by using platescoated with the following respective monoclonal antibodies as controlantibodies instead of the human anti-human AILIM monoclonal antibodies.

(1) Mouse monoclonal antibody SA12 or SG430 against human AILIM (JP-A11-29599 (Example 12) and WO98/38216 (Example 12));

(2) Mouse anti-human CETP monoclonal antibody JHC1 (also referred to asJMab109; JP-A 9-20800); and

(3) Human anti-KLH monoclonal antibody (also referred to as JMab23; theabove-mentioned Example).

The microplates coated with the antibodies were used in the followingassays.

<7-3> Preparation of Human T cell Suspension

Peripheral blood was collected from each normal healthy persons (5persons; donor A, B, C, D and E). Fraction containing mononuclear cellswas prepared by density-gradient centrifugation using LymphoPrep(Nycomed). Human T cells were separated from the human mononuclear cellfraction according to the manual for experimental procedure by using aPan-T cell Isolation Kit (Miltenyi) and Magnetic Sorter. T cell countwas determined using a hemacytometer. Human T cells were suspended inRPMI1640 medium containing 10% FCS to prepare human T cell suspension(1×10⁶ cell/ml).

<7-4> Cell Culture

(1) Culture Using Microplate Coated with Anti-Human CD3 Antibody andAnti-Human AILIM Antibody

Human T cell suspension (donor A, B, C, D or E; 100 μl/well; 1×10⁵cells/well) was added to each well of the microplate coated with theantibody mentioned above and the plate was incubated at 37° C. for 3days in a CO₂ incubator.

After cultivation, aliquots of the resulting culture supernatants (50μl) were stored at −20° C. and then used in the assay described later(assay for IFNγ). After sampling aliquots of the culture supernatants,respective microplates were used for the following assay:

(2) Culture Using Microplate Coated with Anti-Human CD3 Antibody Alone

Human T cell suspension (donor D; 100 μl/well; 1×10⁵ cells/well) wasadded to each well of the microplates coated with the above-mentionedantibody, and then any one of the various human anti-human AILIMmonoclonal antibodies was added thereto (25 μl of the antibody of 40μg/ml-0.0049 μg/ml). The plates were incubated at 37° C. for 3 days in aCO₂ incubator.

<7-5> Determination of Proliferation Activity of T Cell

Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was added toeach well of the plates after incubation, and the plates were incubatedat 37° C. for 6 hours in a CO₂ incubator. After incubation, cells weretrapped on GF/C filters (Packard) using Cell Harvester. Subsequently,the filters were dried at 40° C. for 3 hours or longer, and thenMicroscinti 0 (20 μl/well; Packard) was added thereto. Radioactivity of³H incorporated of the cells trapped on the filters was measured by aβ-counter (TOP COUNT) to analyze the degree of T cell proliferationafter cultivation.

Results are shown in FIGS. 15 to 39.

Result of this assay showed that human T cells were significantlyproliferated depending on the concentration of the cells whenmicroplates were coated with anti-human AILIM monoclonal antibody (humanmonoclonal antibody or mouse monoclonal antibody) together withanti-human CD3 antibody. Further, there were some differences in thedegree of cell proliferation among the donors.

On the other hand, human T cells did not grow significantly when plateshad been coated with anti-human CD3 antibody alone and anti-human AILIMmonoclonal antibody (human monoclonal antibody or mouse monoclonalantibody) in solution (liquid phase) was used during culturing thecells.

<7-6> Quantification of IFNγ in Culture Supernatant of T Cell

For respective cultures of T cells (donors B and C) described in (1) of<7-4>, the amounts of IFNγ in the culture supernatants were determinedby a commercially available human IFNγ ELISA KIT (Amersham-Pharmacia;Endogen).

Results are shown in FIGS. 40 to 47.

Result of this assay showed that the production of IFNγ increasedsignificantly depending on the concentration of anti-human AILIMmonoclonal antibody (human monoclonal antibody or mouse monoclonalantibody).

EXAMPLE 8 Regulatory Activity of Human Anti-Human AILIM MonoclonalAntibody on Mixed Lymphocyte Reaction (MLR)

It was tested whether or not the human anti-human AILIM monoclonalantibodies of the present invention were capable of controlling(enhancing and/or inhibiting) T cell responses (production of cytokinessuch as IFN-γ and IL-4, cell proliferation, etc.), in other words,capable of regulating the transduction of AILIM-mediated costimulatorysignal into cells, by analyzing the activity (namely, DNA synthesis incells) of controlling T cell proliferation associated with allogenicmixed lymphocyte reaction (allogenic MLR) as an index.

<8-1> Preparation of Human PBMC and T Cell

Peripheral blood (200 ml) collected from each normal healthy persons (7persons; donor A, B, C, D, E, F and G) was dispensed on the layers ofLymphoprep (15 ml; Nycomed) in microtubes (50 ml; Falcon). Aftercentrifugation (at 1600 rpm for 10 minutes), intermediate layers wererecovered. Recovered cells were diluted 2 times or further withphosphate buffer, and then centrifuged (at 1,800 rpm for 10 minutes).Thus, PBMC (peripheral blood mononuclear cell; 2×10⁸-5×10⁸ cell) wasprepared. Cell count was determined by using a hemacytometer. An aliquotof the cells to be used in MLR assay (1.08×10⁸ cell/9 microplates) weretaken and kept on ice. Remaining cells were used for the separation of Tcells described below.

PanT Isolation kit (Miltenyi Biotech) was used for the separation of Tcells from PBMC. According to the manual attached to the kit, remainingPBMCs were added to the solution attached to the kit, and the solutionwas incubated. Subsequently, cells were washed with PBS containing 5 mMEDTA and 0.5% BSA and then re-suspended in PBS. Subsequently, the cellsuspension was added to a Positive Selection Column VS+ (MiltenyiBiotech) swollen with PBS, and unadsorbed fraction was recovered.Further, PBS was loaded onto the column, and the wash solution wasrecovered. The same treatment was repeated once. Recovered solutionswere combined together to give a T cell fraction. After centrifugationof the T cell fraction, cells were re-suspended in PBS. Cell count ofthe resulting T cells was determined by using a hemacytometer. The cellswere used in the following assay.

<8-2> Mixed Lymphocyte Reaction (MLR)

As described above, two signaling pathways one between CD28 andCD80(B7-1)/CD86(B7-2) and the other between CTLA4 andCD80(B7-1)/CD86(B7-2), for which comparatively detailed analysis havebeen previously made, are known as costimulatory signaling pathwaysrequired for the activation of lymphocytes such as T cell, etc.

Namely, the proliferation of T cell in response to mixed lymphocytereaction (MLR) can be induced by the signal transduction through each ofthe two known pathways.

Thus, by using the substances indicated below, test of this inventionwas conducted to analyze (1) the inhibition of MLR by blocking theCTLA4-mediated signaling pathway; (2) the inhibition of MLR by blockingthe CD80 (B7-1)/CD86(B7-2)-mediated signaling pathway; (3) theinhibition of MLR by blocking both CTLA4-mediated pathway and CD80(B7-1)/CD86 (B7-2)-mediated signaling pathway; (4) the inhibition of MLRby blocking the tertiary signaling pathway associated with AILIM; and(5) the inhibition of MLR by blocking both CTLA4-mediated pathway andAILIM-mediated pathway.

Following test substances were used.

(1) Human anti-human AILIM monoclonal antibody (prepared in the Exampledescribed above);

(2) Mouse anti-human AILIM monoclonal antibody SA12 (same as in theabove Example);

(3) Human anti-KLH monoclonal antibody (negative control; same as in theabove Example);

(4) Mouse IgG antibody (anti-human CD34; negative control; Immunotech);

(5) A mixture of anti-human CD80 monoclonal antibody (Pharmingen) andanti-human CD86 monoclonal antibody (Pharmingen); and

(6) Human CTLA4-IgFc chimera molecule (Ancell).

Mixed lymphocyte reaction (MLR) was conducted on the following 6combinations using PBMCs and T cells prepared from the donors describedabove in <8-1>.

(i) T cell (donor A)/PBMC (donor D)

(ii) T cell (donor D)/PBMC (donor B)

(iii) T cell (donor C)/PBMC (donor A)

(iv) T cell (donor E)/PBMC (donor G)

(v) T cell (donor F)/PBMC (donor E)

(vi) T cell (donor G)/PBMC (donor F)

The concentrations of PBMCs and T cells to be used in the test wereadjusted as described below.

PBMCs were suspended in PBS, and then transferred into culture dishes(60 mm). The cells were subjected to X-ray irradiation (50 Gy) with anirradiator (Hitachi MEDICO). Cells were recovered, centrifuged and thenadded to PRMI1640 medium containing 10% FCS. Cell count was adjusted to2×10 cells/50 μl.

Resulting T cells from each donor were also added to PRMI1640 mediumcontaining 10% FCS and the cell count was adjusted to 1×10⁵ cells/50 μl.

<8-2-1> Inhibition of MLR by Human Anti-Human AILIM Monoclonal Antibody

PRMI1640 medium containing 10% FCS was added to each well of a 96-wellmicroplate having U-shaped wells. A solution of human anti-human AILIMmonoclonal antibody or mouse anti-human AILIM monoclonal antibody SA12was diluted with PRMI1640 medium containing 10% FCS to prepare solutionswith various concentrations of the antibody. Diluted antibody solutionswere added to the wells (final concentration: 0, 0.31, 1.25, 5 and 20μg/ml). Subsequently, T cells (50 μl) were added to the wells. The platewas incubated at 37° C. for 1 hour in a CO₂ incubator (NAPCO). After thereaction was completed, PBMCs (50 μl) derived from a different donorwere added to the wells to initiate MLR.

When MLR was conducted using an antibody other than human anti-humanAILIM antibody (described above in (3) to (6)) as the test substance, Tcells derived from a different donor were allowed to react after theincubation of PBMCs with the test substance.

On the fifth day of the culture, tritium-labeled thymidine(³H-Thymidine; 20 μl; 1 μCi/well) diluted with PRMI1640 mediumcontaining 10% FCS was added to each well. Cultivation was continued forone day. After the culture was completed, the cells were harvested usinga Cell Harvester (Packard). Radioactivity of ³H incorporated of thecells was measured in a β-counter (TOP COUNT; Packard) to analyze therate of T cell proliferation after the culture.

Results are shown in FIGS. 48 to 59.

<8-2-2> Inhibition of MLR by Human Anti-Human AILIM Monoclonal Antibodyin MLR System Where CTLA4-Mediated Signaling Pathway has been PreviouslyBlocked

PRMI1640 medium containing 10% FCS was added to each well of a 96-wellmicroplate having U-shaped wells. A solution of human anti-human AILIMmonoclonal antibody or mouse anti-human AILIM monoclonal antibody SA12was diluted with PRMI1640 medium containing 10% FCS to prepare solutionswith various concentrations of the antibody. The diluted antibodysolutions were added to the wells (final concentration: 0, 0.31, 1.25, 5and 20 μg/ml). Subsequently, T cells (50 μl) were added to the wells.The plate was incubated at 37° C. for 1 hour in a CO₂ incubator (NAPCO).

In addition to the culture of the T cells, PBMCs (in RPMI1640 mediumcontaining 10% FCS) derived from other donors were culturedindependently after adding human CTLA4-IgFc to the PBMCs. Cultivationwas performed at 37° C. for 1 hour in a CO₂ incubator (NAPCO).Concentration of CTLA4-IgFc was adjusted to 20 μg/ml at the start of theMLR.

Subsequently, PBMCs (50 μl) were added to the T cell culture describedabove to initiate MLR.

When MLR was conducted using an antibody other than human anti-humanAILIM antibody (described above in (3) to (5)) as the test substance, Tcells derived from a different donor were allowed to react after theincubation of PBMCs, which had been cultured in the presence ofCTLA4-IgFc, with the test substance.

On the fifth day of the culture, tritium-labeled thymidine(³H-Thymidine; 20 μl; 1 μCi/well) diluted with PRMI1640 mediumcontaining 10% FCS was added to each well. Cultivation was continued forone day. After the culture was completed, the cells were harvested byusing a Cell Harvester (Packard). Radioactivity of ³H incorporated ofthe cells was measured in a β-counter (TOP COUNT; Packard) to analyzethe rate of T cell proliferation after the culture.

Results are shown in FIGS. 60 to 69.

The results obtained from the two tests described above are summarizedas follows:

(1) CTLA4-IgFc blocks the CTLA-4-mediated signal transduction, andthereby inhibiting the allogenic MLR-induced proliferation of T cell.

(2) Anti-CD80 antibody and anti-CD86 antibody inhibit the signaltransduction mediated by CD80/CD86, which is a ligand for CTLA4 andCD28, and thereby inhibiting the allogenic MLR-induced proliferation ofT cell.

(3) A monoclonal antibody against human AILIM, like CTLA4-IgFc,anti-CD80 antibody and anti-CD86 antibody, significantly inhibits theallogenic MLR-induced T cell proliferation associated with theAILIM-mediated signal transduction in an antibodyconcentration-dependent manner.

In other words, these results show that a tertiary pathway mediated byAILIM and the ligand thereof in addition to the known pathways mediatedby CTLA4/CD80/CD86 and mediated by CD28/CD80/CD86 exist as acostimulatory signaling pathways required for T cell activation, as wellas that the AILIM-mediated signaling pathway is inhibited by antibodyagainst AILIM.

Furthermore, it raises the possibility that contribution ofAILIM-mediated pathway to the signal transduction may be comparable tothose of CTLA4/CD80/CD86-mediated pathway and CD28/CD80/CD86-mediatedpathway.

EXAMPLE 9 Activity of Human Anti-Human AILIM Monoclonal Antibody toInduce Antibody-Dependent Cellular Cytotoxicity (ADCC)

Biological activities caused by antibodies include induction ofantibody-dependent cellular cytotoxicity (ADCC). ADCC is a cytotoxicaction that requires the antibody in addition to effector cells andtarget cells, that induces damage on the target cells induced by theeffector cells such as lymphocyte, macrophage or polymorphonuclearleucocyte.

The activity of the anti-human AILIM monoclonal antibody of the presentinvention to induce ADCC was analyzed as follows:

⁵¹Cr (0.1 mCi/10⁶ cells; Amersham-Pharmacia) was added to the culture ofhuman AILIM-overexpressing recombinant HPB-ALL cells prepared in theExample described above, and the mixture was incubated at 37° C. for 2hours. The cells were washed 8 times with RPMI1640 medium. Theisotope-labeled cells obtained were used as target cells.

Control experiments were performed using wild-type human HPB-ALL cellslabeled with the isotope as control cells in the same manner asdescribed above.

By using Lymphosepar I (IBL), PBMC fractions were separated fromperipheral blood collected from normal healthy persons. The resultinghuman PBMCs were used as effector cells.

The target cells (1×10⁴ cells/well; 25 μl/well) were plated on each wellof a 96-well microplate (Nunc) having U-shaped wells. Subsequently, anyone of various concentrations of human anti-human AILIM monoclonalantibodies diluted with RPMI1640 medium containing 5% FBS (0.0001-1.0μg/ml; 25 μl/well), the medium alone (25 μl/well) or 1% Nonidet P-40 (25μl/well; detergent having cell-lysing activity) was added to each welland the plate was incubated at room temperature for 20 minutes.

Cultivation was carried out using anti-human CD3 monoclonal antibodyOKT3 (ATCC CRL-8001) as a positive control antibody instead ofanti-human AILIM antibody in the same manner as described above.

Subsequently, the effector cells (E/T ratio=50; 1×10⁵ cells/well; 50μl/well) were added to each well and the plate was incubated at 37° C.for 16 hours under an atmosphere of 5% CO₂ in an incubator.

After cultivation, samples were centrifuged (at 1,500 rpm at 4° C. for10 minutes). Resulting supernatant was recovered. Radioactivity in thecentrifugal supernatant was measured by a γ-counter. The radioactivityrepresents the amount of ⁵¹Cr released from the cells into the culturesupernatant by the damage of cell membrane by ADCC.

Percentage of cell membrane damage (percentage cell lysis), which wascaused by ADCC induced by anti-AILIM antibody or anti-CD3 antibody, wasdetermined under an assumption that the radioactivity observed with themedium alone corresponds to 0% with respect to the cell membrane damage(0) and that with Nonidet corresponds to 100% with respect to the cellmembrane damage.

Results are shown in FIGS. 70 and 71.

The result of the test showed that human anti-human AILIM monoclonalantibody of the present invention exhibited ADCC-inducing activity in aconcentration-dependent manner.

EXAMPLE 10 Determination of Gene and Amino Acid Sequences of HumanAnti-Human AILIM Monoclonal Antibody and Analysis of the Same

Sequences of cDNAs encoding the heavy chains as well as cDNAs encodingthe light chains of various human anti-human AILIM monoclonalantibodies, which had been prepared in the Example described above, weredetermined as described below. Structural features of the genes werealso analyzed.

By using Quick Prep mRNA Purification Kit (Amersham-Pharmacia),PolyA⁺RNAs were extracted and purified from each of hybridomas (clones:AIH5D3 (JMab-136), AII289 (JMab-138) and AII394 (JMab-139)), whichproduce human monoclonal antibody against human AILIM prepared in theExample described above.

The hybridoma cells were suspended in a cell lysis buffer (LysisBuffer), and lysed by using a syringe to solubilize them. Oligo (dT)resin was added to the solubilized material and the mixture was shakengently. Subsequently, Oligo (dT) resin was washed, and then PolyA⁺RNAwas eluted with Elution Buffer. Eluted PolyA⁺RNA was precipitated withethanol, and then dissolved in Tris-EDTA buffer. Concentration ofPolyA+RNA obtained was determined by absorbance at a wavelength of 260nm.

Double-stranded cDNA was synthesized by using PolyA⁺RNA as a templateaccording to M-MLV Reverse Transcriptase method using a commerciallyavailable cDNA synthesis kit (GIBCOBRL) and synthetic oligo DNA NotI-T(SEQ ID NO:1) as a primer.

Specifically, single-stranded cDNA was synthesized in a solution (about50 μl) containing PolyA⁺RNA (about 5 μg) purified from the hybridomas asa template, the primer (about 400 pmole) and M-MLV Reverse Transcriptaseat 37° C. for 1 hour. Subsequently, dNTP, DNA polymerase I, RNaseH, DNAligase, buffer and distilled water were added to the reaction solution(4 μl), and the mixture was incubated at 16° C. for 2 hours tosynthesize double-stranded cDNA. The resulting double-stranded cDNA wasextracted with phenol/chloroform and then precipitated with ethanol.

Subsequently, EcoRI linker DNA (about 300 pmole) and DNA ligase(Ligation High; 33 μl; TOYOBO) was added to the solution containing thedouble-stranded cDNA in TE buffer (about 50 μl) and the mixture wasincubated at 16° C. for about 80 minutes to ligate the cDNA with thelinker DNA. The linker DNA used was a double-stranded DNA consisting ofoligo DNA (20adp; SEQ ID NO:2) and oligo DNA (24adp; SEQ ID NO:3), whichhad been 5′-phosphorylated and annealed to each other by a commonly usedmethod.

The DNA ligate was extracted with phenol/chloroform, and thenprecipitated with ethanol. Subsequently, the DNA reactant was digestedwith a commercially available restriction enzyme NotI (TOYOBO), and thenincubated with a commercially available ATP solution (GIBCO BRL) and T4kinase (TOYOBO) at 37° C. for 30 minutes to phosphorylate the 5′ endthereof.

The resulting DNA was precipitated with ethanol-, and then fractionatedby polyacrylamide gel electrophoresis. A piece of gel containing DNA ofabout 500 bp to 2000 bp was cut out. Cutting of the gel was carried outwhile the DNA stained with ethidium bromide was being visualized byirradiating UV light in a photographic device.

The gel cut off was crushed and then suspended in TE buffer. Thesuspension was centrifuged and the resulting supernatant was recovered.

The DNA recovered was ligated to a commercially available lambda phagevector λEXcell (0.25 μg; Amersham Pharmacia) in the presence ofcommercially available DNA Ligase (Ligation High; TOYOBO) (at 16° C. for30 minutes). In the next step, the DNA ligate was packaged into lambdaphage using a commercially available lambda phage packaging kit GigapackIII Gold (STRATAGENE) and the resulting phage particles were infected toE. coli NM522 as a host to prepare a cDNA library. All the manipulationswere carried out according to the experimental protocol attached to thekit.

Subsequently, the cDNA library was screened by a plaque hybridizationmethod (Maniatis et al., “Molecular Cloning: A Labolatory Manual,” ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.) as follows:

The cDNA library (1×10⁴ plaques) was plated on agar plates and replicafilters thereof were prepared by using Hybond-N nylon membranes(Amersham Pharmacia). These replica filters were subjected tohybridization treatment using probes labeled by using γ³²P-ATP in ahybridization buffer according to the plaque hybridization method.Probes used were HIGLC (SEQ ID NO:4) for antibody light chain and NHCc2(SEQ ID NO:5) for antibody heavy chain. Single-plaque isolation wascarried out from the positive clones obtained in the primary screeningand secondary screening.

Each of heavy chain and light chain of the antibody was amplified by PCRusing a single PCR primer and Taq PCR kit (TAKARA) by utilizing phagesuspension from each positive clone as a template DNA. A pair of primersused for antibody light chain were ExcellE (SEQ ID NO:6) and ck117 (SEQID NO:7), and a pair of primers used for antibody heavy chain wereExcellE (SEQ ID NO:6) and NHCc2 (SEQ ID NO:5). The resulting PCRproducts were fractionated according to a usual method using agarose gelelectrophoresis. Pieces of gel containing DNAs of about 600 bpcorresponding to the heavy chain and light chain were cut out.Nucleotide sequences of the DNAs purified from the gel were analyzed byusing a DNA Sequencer (373A; PE-Applied Biosystems), ABI PRISMSequencing Software (PE-Applied Biosystems) and ABI PRISM Auto Assembler(PE-Applied Biosystems). DNA from each positive clone was verified tohave sufficient length of nucleotide sequence.

λPhage from the plaque of each positive clone was infected to E. coliNP66 for in vivo excision of plasmid DNA of interest, and the resultingfilamentous phages were plated on ampicillin-containing plates to givecolonies. Subsequently, plasmid DNAs were recovered and purified fromthe colonies by a commonly used method, and E. coli JM109 wastransformed with the plasmids. Subsequently, the transformed cells wereplated on ampicillin-containing nutrient agar plates to form colonies.

Subsequently, bacterial suspension in ampicillin-containing LB mediumderived from each colony was transferred to a liquid nutrient medium andthe bacteria were cultured at 37° C. for 24 hours. The bacteria wereharvested from the culture, and then the plasmid DNA was purified by aplasmid purification kit (Quiagen). Each of the plasmid DNAs wasdigested with restriction enzymes EcoRI/NotI to verify the presence ofvector DNA and insert DNA (heavy chain cDNA or light chain cDNA).

Each nucleotide sequence of cDNA encoding heavy chain and antibody lightchain of the antibody, which was inserted in each purified plasmid, wasdetermined by a commonly used method using DNA Sequencer (377A;PE-Applied Biosystems), ABI PRISM Sequencing Software (PE-AppliedBiosystems) and ABI PRISM Auto Assembler (PE-Applied Biosystems).

Primers used for the sequence determination were as follows:

<Primers Used for the Determination of Heavy Chain cDNA>

M13R primer (SEQ ID NO:8; STRATAGENE), ExcellE (SEQ ID NO:6), 136H (SEQID NO:9), 138/9H (SEQ ID NO: 10), AILIMHC1 (SEQ ID NO: 11), HCc1 (SEQ IDNO: 12), NHCc2 (SEQ ID NO:5), HCc7 (SEQ ID NO: 13), HCc8 (SEQ ID NO:14), HCc3 (SEQ ID NO:15), HCc4 (SEQ ID NO:16), HCc6 (SEQ ID NO: 17),HIGHC (SEQ ID NO:18), HCc9 (SEQ ID NO:19), HCc5 (SEQ ID NO:20) and polyA(SEQ ID NO:21).

<Primers Used for the Determination of Light Chain cDNA>

M13R primer (SEQ ID NO:8; STRATAGENE), ExcellE (SEQ ID NO:6), AILIMLC1(SEQ ID NO:22), AILIMLC2 (SEQ ID NO:23), LCc1 (SEQ ID NO:24), ck117 (SEQID NO:7), HIGLC (SEQ ID NO:4), LCc2 (SEQ ID NO:25), HIK (SEQ ID NO:26),and polyA (SEQ ID NO:21).

Sequence Listing shown below contains cDNA sequence encoding heavy chainand cDNA sequence encoding light chain of human monoclonal antibodyagainst human AILIM, which are produced by each hybridoma mentionedabove, as well as amino acid sequences deduced from the cDNA sequences.

Clone AIH5D3 (JMab-136) <Heavy Chain>

DNA sequence: SEQ ID NO:27 (signal sequence: nucleotide number 69 to125, V region: nucleotide number 126 to 419)

Amino acid sequence: SEQ ID NO:28 (comprising signal sequence: aminoacid number 1 to 19, variable region: amino acid number 20 to 118)

<Light Chain>

DNA sequence: SEQ ID NO:29 (signal sequence: nucleotide number 39 to104, V region: nucleotide number 105 to 386)

Amino acid sequence: SEQ ID NO:30 (comprising signal sequence: aminoacid number 1 to 22, variable region: amino acid number 23 to 116)

Clone AII289 (JMab-138) <Heavy Chain>

DNA sequence: SEQ ID NO:31 (comprising signal sequence: nucleotidenumber 94 to 150, V region: nucleotide number 151 to 441)

Amino acid sequence: SEQ ID NO:32 (comprising signal sequence: aminoacid number 1 to 19, variable region: amino acid number 20 to 116)

<Light Chain>

DNA sequence: SEQ ID NO:33 (comprising signal sequence: nucleotidenumber 28 to 87, V region: nucleotide number 88 to 375)

Amino acid sequence: SEQ ID NO:34 (comprising signal sequence: aminoacid number 1 to 20, variable region: amino acid number 21 to 116)

Clone AII394 (JMab-139) <Heavy Chain>

DNA sequence: SEQ ID NO:35 (signal sequence: nucleotide number 96 to152, V region: nucleotide number 153 to 443)

Amino acid sequence: SEQ ID NO:36 (comprising signal sequence: aminoacid number 1 to 19, variable region: amino acid number 20 to 116)

<Light Chain>

DNA sequence: SEQ ID NO:37 (signal sequence: nucleotide number 33 to 92,V region: nucleotide number 93 to 380)

Amino acid sequence: SEQ ID NO:38 (comprising signal sequence: aminoacid number 1 to 20, variable region: amino acid number 21 to 116)

By using analytical software for gene sequence, the library V BASESequence for human immunoglobulin variable region genes constructed byTomlinson et al. (Immunol. Today, Vol. 16, No. 5, p. 237-242, 1995) wassearched for each of the DNA sequences determined herein.

Result showed that the V-region genes of the respective heavy chain andlight chain of the above-mentioned human monoclonal antibodies consistedof the following segments.

<Heavy Chain V-Region Gene>

clone AIH5D3 (JMab-136): 1-02

clone AII289 (JMab-138): 3-13

clone AII394 (JMab-139): 3-13

<Light Chain V-Region Gene>

clone AIH5D3 (JMab-136): L5

clone AII289 (JMab-138): A27

clone AII394 (JMab-139): A27

EXAMPLE 11 Inhibitory Effect of Human Anti-Human AILIM MonoclonalAntibody on Delayed-Type Hypersensitivity (DTH)

The biological system of immune response, the function of which is toeliminate harmful antigens (pathogenic microorganisms such as virus,bacterium and parasite, foreign body, etc.) to the living bodies, andcan be broadly divided into congenital immunity and acquired immunity.

The former is a system of elimination based on non-specific recognitionincluding phagocytosis by phagocytes (polymorphonuclear leukocyte,monocyte, macrophage, etc.), attack by natural killer (NK) cells, andopsonization of antigen by complement, etc.

The latter, acquired immune response, is a system of elimination bylymphocytes (mainly, T cell and B cell) which acquired specificity(activation) to the antigen.

Further, acquired immune response can broadly be divided into cellularimmunity and humoral immunity.

Unlike antibody-dependent humoral immunity, cellular immunity is animmune response expressed in general by the direct action of T cell onan antigen as the target. Cellular immunity is known to be involved inimmune response to virus or tumor, immune response induced aftertransplantation of tissue or organ, hypersensitivity to some drugs, andsome of autoimmune diseases.

Most typical phenomenon belonging to cellular immunity is the well-knowntuberculin allergy (almost synonymous with tuberculin hypersensitivity).Tuberculin allergy is a delayed allergy triggered by the infection bytubercle bacillus. The allergy is due to the infection by tuberclebacillus and can be induced by causing immune response byintracutaneously injecting, to a living body, tuberculin proteinproduced in culture supernatant of tubercle bacillus.

Delayed allergy is an allergy mediated by T cell (memory T cellmemorizing antigen) sensitized with an antigen. The allergy is called“delayed type,” because it takes 24 to 48 hours for a living bodysensitized with the antigen to express allergic response withinflammation induced by the memory T cell when contacted again with theantigen.

The phenomenon of tuberculin allergy, which is a representative ofdelayed allergies, has generally been utilized in “tuberculin test” todiagnose whether or not sensitization by the infection of tuberclebacillus has already been established in an animal. Specifically, thetest is conducted as follows: purified tuberculin (purified proteinderivative; PPD; 0.1 ml of the derivative of 0.05 μg/0.1 ml (2.5 TU) forgeneral diagnostic use), which is tuberculin protein purified from theculture of tubercle bacillus, is intracutaneously given to an animal;the major axis of skin redness at the injection site is measured 48hours after the injection; and the presence of infection of tuberclebacillus can be diagnosed based on the major axis measured. If the majoraxis of the redness is shorter than 4 mm, then the subject is negative;if it is within the range of 4-9 mm then the subject is false positive;and if it is 10 mm or longer then the subject is decided positive.

Delayed allergies associated with cellular immunity include, forexample, Jones-Mote type hypersensitivity transiently induced by a smallamount of protein or the like, contact allergy to drugs such as picrylchloride or plant toxins such as Japanese lacquer, or allergy associatedwith graft rejection (e.g., allogenic graft) as well as theabove-mentioned allergy to antigen from infectious pathogen such astuberculin allergy caused by tubercle bacillus described above.

In this test, the inhibitory effect of anti-AILIM antibody ondelayed-type hypersensitivity (delayed allergy) was evaluated byutilizing the above-mentioned tuberculin test. The test was conducted asfollows:

Each of cynomolgus monkeys (male, body weight: 6.0-8.5 kg, EnvironmentalBiological Life Science Research Center Inc.; 3 individuals in eachgroup), which had been sensitized with BCG (Bacille de Calmette etGuerin), which is an attenuated live bacterium of bovine-type tuberclebacillus, was anesthetized with ketamine hydrochloride (10 mg/kg,intramuscular injection), and then any one of the test samples indicatedbelow was intracutaneously given with a quantity of 0.1 ml to eachinjection site (6 injection sites/individual) in the chest. Thedistances between the injection sites of the sample were 3 cm or longer.

(1) 1:1 mixed solution of human anti-human AILIM monoclonal antibody(JMab-136; 0.2 mg; 10 μg at each injection site) and tuberculin (4 μg/1ml of physiological saline);

(2) 1:1 mixed solution of human anti-human AILIM monoclonal antibody(JMab-136; 2 mg; 100 μg at each injection site) and tuberculin (4 μg/1ml of physiological saline);

(3) phosphate buffer (PBS (−)) as a control;

(4) 1:1 mixed solution of a commercially available steroidalanti-inflammatory agent, Prednisolone (0.2 mg; 10 μg at each injectionsite) and tuberculin (4 μg/1 ml of physiological saline) as a positivecontrol;

(5) 1:1 mixed solution of human anti-KLH monoclonal antibody (Example<2-2>; 0.2 mg; 10 μg at each injection site) and tuberculin (4 μg/1 mlof physiological saline) as a negative control.

24 hours after injection of each sample, the major axis and minor axisof redness at each injection site were measured to determine the area ofthe redness.

The result is shown in FIG. 72.

The result showed that redness due to delayed allergy was significantlyinhibited in any groups subjected to injection of the anti-AILIMantibodies, as compared with the control and negative control groups.The inhibitory effect was comparable to that of the steroidalanti-inflammatory drug used as a positive control.

EXAMPLE 12 Analysis for the Expression of AILIM in Various Tissues ofPatients with Graft Versus Host Disease (GVHD)

Expression of AILIM in a variety of tissues obtained of biopsy frompatients, who were recipients subjected to transplantation of allogenicgraft from donors and had been diagnosed clinically to be affected withacute or chronic graft versus host disease (GVHD) after thetransplantation, was analyzed by a commonly used method. Tissues werestained with HE and human anti-human AILIM monoclonal antibody (JMab-36)prepared in the Example described above.

Analysis was carried out using 33 samples form various tissues collectedfrom acute GVHD patients (28 cases) as well as 5 samples from chronicGVHD patient (5 cases).

Results were as follows: AILIM-positive reaction was found in 15 of 29samples of skin tissue; in 1 of 3 samples of stomach tissue; and in 1 of1 sample of colon tissue; which were all obtained from acute GVHDpatients. AILIM-positive reaction was found in 1 of 3 samples of skintissue; in 2 of 2 samples of colon tissue; which were all obtained fromchronic GVHD patients. Further, AILIM-positive reaction was found in 10of 13 samples in which significant lymphocyte infiltration had beenobserved.

EXAMPLE 13 Activity of Human Anti-Human AILIM Monoclonal Antibody toTransduce Costimulatory Signal in Monkey T Cell

The experiment conducted in Example 7 has demonstrated that the humananti-human AILIM monoclonal antibodies of the present invention arecapable of enhancing the proliferation of human T cell via controllingthe human T cell response, specifically, controlling the transduction ofAILIM-mediated costimulatory signal to the cell. In this test, it wasanalyzed whether or not the human monoclonal antibodies exhibit activityof enhancing cell proliferation of monkey T cell by the same method asdescribed in Example 7.

<13-1> Dilution of Antibody

Anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) was diluted withphosphate buffer (PBS) to a final concentration of 1 μg/ml.

The above-prepared human anti-human AILIM monoclonal antibody JMAb136was diluted with PBS to a final concentration of 40 μg/ml. The antibodysolutions were further diluted with PBS to prepare variousconcentrations of antibodies (40 μg/ml-0.064 μg/ml).

<13-2> Coating of Microplate with Antibody

Each well of 96-well microplates was coated with anti-human CD3monoclonal antibody SP34 (1 μg/ml; 25 μl to each well) and the humananti-human AILIM monoclonal antibody JMAb136 (40 μg/ml-0.064 μg/ml; 25μl to each well). The plates were incubated at 37° C. for 2 hours.Subsequently, the antibody solutions were discarded, and each well waswashed 3 times with PBS. After the wash, RPMI1640 medium containing 10%FCS was added to each well (100 μl/well), and the plates were incubatedat 37° C. for 1 hour. Thus respective wells of the plates were coatedwith the antibodies mentioned above.

Control experiments were carried out in the same manner using platescoated with human anti-KLH monoclonal antibody (JMab23; see the previousExamples) as control antibodies instead of the human anti-human AILIMmonoclonal antibodies.

The microplates coated with the antibodies were used in the followingassays.

<13-3> Preparation of Monkey T Cell Suspension

Peripheral blood was collected from cynomolgus monkeys and mononuclearcells were fractionated by density gradient centrifugation usingNycoPrep1.077A (Nycomed). According to the experimental manual, monkey Tcells were separated from the cynomolgus monkey mononuclear cells byusing anti-human CD4 antibody M-T477 (BD-Pharmingen), anti-human CD8antibody RPA-T8 (BD-Pharmingen), anti-mouse IgG microbead (Miltenyi) anda Magnetic Sorter. T cell count was determined using a hemacytometer.Monkey T cells were suspended in RPMI1640 medium containing 10% FCS.Thus monkey T cell suspension (1×10⁶ cell/ml) was prepared.

<13-4> Cell Culture

(1) Culture Using Microplate Coated with Anti-Human CD3 Antibody andAnti-Human AILIM Antibody

Simian T cell suspension was added to each well of a microplate coatedwith the antibody mentioned above and the plate was incubated at 37° C.for 2 days in a CO₂ incubator.

After the culture was completed, respective microplates were used in thefollowing assays:

<13-5> Determination of Proliferation Activity of T Cell

Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was added toeach well of the plates after incubation, and the plates were incubatedat 37° C. for 6 hours in a CO₂ incubator. After incubation, the cellswere trapped on GF/C filters (Packard) by using a Cell Harvester.Subsequently, the filters were dried at 40° C. for 3 hours or longer,and then Microscinti 0 (20 μl/well; Packard) was added thereto.Radioactivity of ³H incorporated in the cells trapped on the filters wasmeasured by a β-counter (TOP COUNT) to analyze the degree of T cellproliferation after the culture.

The result is shown in FIG. 73.

The result of this assay showed that simian T cells were significantlyproliferated depending on the concentration of the cells whenmicroplates were coated with anti-human AILIM monoclonal antibody (humanmonoclonal antibody or mouse monoclonal antibody) together withanti-human CD3 antibody.

The result also suggests that the human anti-human AILIM monoclonalantibodies of the present invention can bind to monkey AILIM and havethe activity to regulate the function of monkey AILIM.

EXAMPLE 14 Establishment of Method for Identifying and QuantifyingSubstances Capable of Binding to AILIM or AILIM Ligand

A method of ELISA (Enzyme-linked Immuno solvent assay) was establishedto identify or quantify a substance capable of binding to AILIM (ICOS)or AILIM ligand (B7h/B7RP1/GL50/LICOS).

The principle of the method described below in detail as an example isbased on estimating, by ELISA, the degree of inhibition on the bindingbetween soluble human AILIM (hAILIM-IgFc) and soluble human AILIM ligand(hB7h-IgFc) caused by the substance.

<14-1> Sample

The following samples were used:

(1) Streptavidin-HRP (Southern Biotechnology Associates, Inc.);

(2) Soluble human AILIM ligand (fusion protein between the extracellularregion of human B7h and the constant region of human IgG1);

The protein was prepared by the method described below in <14-2>;

(3) Biotin-labeled soluble AILIM-IgFc;

The AILIM-IgFc was prepared by further purifying the antigen obtainedaccording to the same method as described in earlier applications (JP-A11-29599 (Example 16 (2)) and WO98/38216 (Example 16 (2))) of one of thepresent inventors, Tezuka;

(4) Human anti-human AILIM monoclonal antibody (JMab-136; describedabove);

(5) Human anti-KLH monoclonal antibody (negative control antibody;JMab-23; described above);

(6) Phosphate buffer (PBS (−); Nikken Seibutsu);

(7) PRMI1640 medium (Nikken Seibutsu);

(8) Fetal calf serum (FCS; JRH-Bioscience);

(9) 30% Bovine serum albumin (BSA; Sigma);

(10) Tween20 (BioRad);

(11) TMB⁺ substrate chromogen (Dako).

<14-2> Preparation of Soluble Human AILIM Ligand (Fusion Protein(hB7h-IgFc) of the Extracellular Region of Human B7h and the ConstantRegion of Human IgG1)

Total RNA was prepared from human peripheral blood-derived mononuclearcells in the same manner as described in the Example above.

cDNA was synthesized from the obtained total RNA (5 μg) as a templateand by using Superscript Preamplification System for First Strand cDNASynthesis (GIBCO-BRL).

Subsequently, 5′primer (5′-GAGGTCTCCGCCCTCGAGATGCGGCTGGGCAGTCC-3′, SEQID NO:39) having XhoI restriction site and 3′primer(5′-CACAGGACAGCCAGGGGATCCCACGTGGCCGCG-3′, SEQ ID NO:40) having BamHIrestriction site at their respective ends were designed and synthesizedto amplify cDNA encoding the extracellular region of human AILIM ligand(hB7h) by PCR. By using the cDNA as a template and the primer pair, PCRwas conducted to prepare a DNA having XhoI and BamHI at respective endsthereof containing cDNA encoding the extracellular region of human B7h.The resulting PCR products were digested with XhoI and BamHI, andfractioned by agarose gel electrophoresis to isolate a bandcorresponding to about 720-bp cDNA fragment, that was predicted toencode the extracellular region of interest. The isolated cDNA fragmentwas subcloned into plasmid pBluescript II SK (+) (Stratagene)pre-digested with XhoI and BamHI. It was verified that the cDNA fragmentcontained the portion encoding the extracellular region of human B7h bysequencing analysis using an automated fluorometric DNA sequencer(Applied Biosystems).

On the other hand, DNA encoding Fc of human IgG1 as a fusion partner wasprepared as a BamHI-XbaI DNA fragment (about 1.3 kb) by digesting aplasmid (see, Cell, Vol. 61, p. 1303-1313, 1990; prepared by Dr. Seed etal., at the Massachusetts General Hospital) with BamHI and XbaI. Thisfragment contained exons encoding the hinge regions of human IgG1, Cγ12,and Cγ13.

The XhoI-BamHI fragment encoding the extracellular region of human B7h(hB7h), and the BamHI-XbaI fragment containing exons encoding Fc(abbreviated as “IgFc”) of human IgG1, prepared as described above, weresubcloned into a plasmid pBluescript II SK (+) (Stratagene) pre-digestedwith XhoI and XbaI.

Subsequently, the plasmid was digested with XhoI and XbaI to prepare aDNA fragment about 1.8-kb containing fusion DNA between theextracellular region of human B7h and human IgFc. By using T4 DNAligase, this fusion DNA fragment was inserted into an expression vectorpME18S (Medical Immunology, Vol. 20, No. 1, p. 27-32, 1990, and“Handbook for Genetic Engineering,” Experimental Medicine, supplement,Yodosha, pp. 101-107, 1992) between XhoI and XbaI sites, to construct aplasmid phB7h-IgFc.

Monolayer COS7 cells (ATCC CRL-1651) cultured to be sub-confluent inDMEM medium containing 10% fetal calf serum and ampicillin, weretransformed with the plasmid phB7h-IgFc by electroporation to yieldtransformed cells.

The transformed cells were allowed to expresshB7h-IgFc by culturing themin serum-free ASF104 medium for 72 hours.

HB7h-IgFc was purified by using a Protein G Sepharose affinity column(Amersham Pharmacia) as follows:

The above-mentioned culture supernatant was centrifuged to obtaincentrifugal supernatant. The resulting supernatant was loaded onto aProtein G Sepharose affinity column pre-equilibrated with a bindingbuffer. Subsequently, the column was washed with the binding buffer, andthen elution was performed with an elution buffer. The eluted solutionwas recovered and then dialyzed against phosphate buffer. The outerdialyzing buffer was changed twice or more. Thus purified hB7h-IgFc wasobtained.

<14-3> Dilution of Antibody and Soluble Human AILIM (□AILIM-IgFc) andreaction Thereof

Original solutions (20 μg/ml) of anti-human AILIM monoclonal antibody(JMab-136) and human anti-KLH monoclonal antibody (JMab-23) as anegative control antibody were diluted in a series of 11 levels, andeach sample (200 μl) was combined and mixed well with 200 μl of RPMI1640medium containing 10% FCS. Thus various concentrations of antibodysolutions were prepared. Biotin-labeled hAILIM-IgFc (2 μl/tube; finalconcentration 1 μg/ml) was added to each of the prepared solutions withvarious concentrations. The resulting solutions were mixed well andincubated at room temperature for 30 minutes.

<14-4> Assay for the Activity of Anti-AILIM Monoclonal Antibody toInhibit the Binding Between hAILIM-IgFc and hB7h-IgFc

hB7h-IgFc was added to each well of a 96-well microplate (50 μl/well(800 ng/well)). The plate was sealed and then incubated at 37° C. for 1hour. Solution was removed from each well, and the wells were washed 3times with PBS (120 μl). Subsequently, PBS containing 0.5% BSA (100μl/well) was added to each well to block the unreacted sites. The platewas sealed and incubated at 37° C. for 1 hour. After incubation, thesolution was removed, and then the wells were washed 3 times with PBS(120 μl). Subsequently, each sample (50 μl/well) prepared in <14-3> wasadded to the wells. The plate was sealed and incubated at 37° C. for 1hour. Solution was removed from each well. The wells were washed 3 timeswith RPMI1640 medium containing 10% FCS (120 μl) pre-heated at 37° C.Subsequently, PBS containing 3.7% formalin (100 μl/well) was added toeach well, and the plate was incubated on ice for 5 minutes. Solutionwas removed from each well, and the wells were washed 3 times with 0.1%Tween20 (120 μl). Subsequently, Streptavidin-HRP (50 μl/well) was addedto each well. The plate was sealed and incubated at room temperature for30 minutes. Solution was removed from each well, and the plate waswashed 3 times with PBS containing 0.1% Tween20 (120 μl). Subsequently,TMB⁺ substrate chromogen (50 μl/well) was added to each well and theplate was incubated at room temperature for 20 minutes. Subsequently, 2Nsulfuric acid (50 μl/well) was added to each well to stop the reaction.Absorbance of each well was measured at a wavelength of 450 nm by aTHERMO max (Molecular Devices).

Results are shown in FIGS. 74 to 76.

The results showed that anti-AILIM antibody had the activity ofinhibiting the binding between hAILIM-IgFc and hB7h-IgFc in adosage-dependent manner.

Accordingly, this Example indicates that an assay system illustrated bythe present method can be utilized to screen and identify substancescapable of binding to AILIM or AILIM ligand (for example, antibody orsynthetic low molecular weight compound).

EXAMPLE 15 Activity of Human Anti-Human AILIM Monoclonal Antibody toInhibit the Proliferation of Human T Cell Associated with theTransduction of AILIM-AILIM Ligand-Mediated Costimulatory Signal

It was analyzed whether or not the anti-human AILIM monoclonalantibodies of the present invention had regulating activity on thetransduction of signal mediated by AILIM on the surface of human T cell,based on the measurement of inhibitory effect of the human anti-humanAILIM monoclonal antibody on cell proliferation induced by contactinghuman T cell with AILIM ligand (B7h/B7RP1/GL50/LICOS).

<15-1> Dilution of Antibody

Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted withphosphate buffer (PBS) to a final concentration of 8 μg/ml.

The soluble human AILIM ligand (hB7h-lgFc) prepared above was dilutedwith PBS to a final concentration of 40 μg/ml. The antibody solutionswere further diluted with PBS to prepare various concentrations ofantibodies (40 μg/ml-0.064 μg/ml).

<15-2> Coating of Microplate with Antibody

Each well of 96-well microplates was coated with (1) anti-human CD3monoclonal antibody OKT3 (8 μg/ml; 25 μl in each well) and hB7h-lgFc (40μg/ml-0.064 μg/ml; 25 μl in each well). The plates were incubated at 37°C. for 2 hours. Subsequently, the antibody solutions were discarded, andeach well was washed 3 times with PBS. After the wash, RPMI1640 mediumcontaining 10% FCS was added to each well (100 μl/well), and the plateswere incubated at 37° C. for 1 hour. Thus respective wells of the plateswere coated.

<15-3> Preparation of Human T Cell Suspension

Peripheral blood was collected from normal healthy persons and themononuclear cells were fractionated by density-gradient centrifugationusing LymphoPrep (Nycomed). According to the experimental manual, humanT cells were separated from the human mononuclear cells by using a Pan-Tcell Isolation Kit (Miltenyi) and a Magnetic Sorter. T cell count wasdetermined by using a hemacytometer. Human T cells were suspended in aRPMI1640 medium containing 10% FCS supplied with human anti-human AILIMmonoclonal antibody JMab136 (20 μg/ml). Human T cell suspension (1×10⁶cells/ml) prepared was incubated at room temperature for 30 minutes.

Human anti-KLH monoclonal antibody JMAb23 (20 μg/ml) was used as anegative control antibody.

<15-4> Cell Culture

In the same manner as described above, human T cell suspension (100μl/well; 1×10⁵ cells/well) was added to each well of a microplate coatedwith anti-human CD3 antibody and hB7h-IgFc, and the plate was incubatedat 37° C. for 3 days in a CO₂ incubator.

<15-5> Determination of Proliferation Activity of T Cell

Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was added toeach well of the plates after cultivation, and the plates were incubatedat 37° C. for 6 hours in a CO₂ incubator. After incubation, the cellswere trapped on GF/C filters (Packard) by using a Cell Harvester.Subsequently, the filters were dried at 40° C. for 3 hours or longer,and then Microscinti 0 (20 μl/well; Packard) was added thereto.Radioactivity of ³H incorporated in the cells trapped on the filters wasmeasured by a β-counter (TOP COUNT) to analyze the degree of T cellproliferation after the culture.

The result is shown in FIG. 77.

The result obtained in this test showed that human T cells grewsignificantly depending on the concentration of human B7h-IgFc (in theassay using the negative control antibody). In addition, the anti-humanAILIM monoclonal antibody significantly inhibited the proliferation ofhuman T cells.

EXAMPLE 16 Activity of Human Anti-Human AILIM Monoclonal Antibody toInhibit the Proliferation of Monkey T Cell Associated with theTransduction of AILIM-AILIM Ligand-Mediated Costimulatory Signal

The same test was conducted by using monkey T cells instead of human Tcells used in Example 15 described above.

<16-1> Dilution of Antibody and Others

Anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) was diluted withphosphate buffer (PBS) to a final concentration of 1 μg/ml.

The human B7h-IgFc prepared above was diluted with PBS to a finalconcentration of 40 μg/ml. The antibody solution was further dilutedwith PBS to prepare various concentrations of the antibody (40μg/ml-0.0064 μg/ml).

<16-2> Coating of Microplate with Antibody

Each well of 96-well microplates was coated with (1) anti-human CD3monoclonal antibody SP34 (BD-Pharmingen) (1 μg/ml; 25 μl in each well)and human B7h-IgFc (40 μg/ml-0.0064 μg/ml; 25 μl in each well). Theplates were incubated at 37° C. for 2 hours. Subsequently, the antibodysolution was discarded, and each well was washed 3 times with PBS. Afterthe wash, RPMI1640 medium containing 10% FCS was added to each well (100μl/well), and the plates were incubated at 37° C. for 1 hour. Thus,respective wells of the plates were coated with the antibody.

<16-3> Preparation of Monkey T Cell Suspension

Peripheral blood was collected from cynomolgus monkeys. A fractioncontaining mononuclear cells was prepared by density-gradientcentrifugation using NycoPrep1.077A (Nycomed). According to theexperimental manual, monkey T cells were separated from the cynomolgusmonkey mononuclear cells by using anti-human CD4 antibody M-T477(BD-Pharmingen), anti-human CD8 antibody RPA-T8 (BD-Pharmingen),anti-mouse IgG microbead (Miltenyi) and a Magnetic Sorter. T cell countwas determined by using a hemacytometer. Monkey T cells were suspendedin RPMI1640 medium containing human anti-human AILIM monoclonal antibodyJMab-136 (20 μg/ml) and 10% FCS to prepare monkey T cell suspension(1×10⁶ cells/ml). The suspension was incubated at room temperature for30 minutes.

Human anti-KLH monoclonal antibody JMab-23 (20 μg/ml) was used as anegative control antibody.

<16-4> Cell Culture

In the same manner as described above, monkey T cell suspension (100μL/well; 1×10⁵ cells/well) was added to each well of a microplate coatedwith anti-human CD3 antibody and hB7h-IgFc, and the plate was incubatedat 37° C. for 3 days in a CO₂ incubator.

<16-5> Determination of Activity of T Cell Proliferation

Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was added toeach well of the plates after the cultivation, and the plates wereincubated at 37° C. for 6 hours in a CO₂ incubator. After theincubation, the cells were trapped on GF/C filters (Packard) by using aCell Harvester. Subsequently, the filters were dried at 40° C. for 3hours or longer, and then Microscinti 0 (20 μl/well; Packard) was addedthereto. Radioactivity of ³H incorporated into the cells trapped on thefilters was measured by a b-counter (TOP COUNT) to analyze the degree ofT cell proliferation after the culture.

The result is shown in FIG. 78.

The result obtained in this test showed that monkey T cells grewsignificantly depending on the concentration of human B7h-IgFc (in theassay using the negative control antibody). In addition, the anti-humanAILIM monoclonal antibody significantly inhibited the proliferationmonkey T cells.

Sequence Listing Free Text SEQ ID NO:1

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, NotI-T.

SEQ ID NO:2

Other Information: Description of Artificial Sequence: Artificiallysynthesized linker sequence, 20adp

SEQ ID NO:3

Other Information: Description of Artificial Sequence: Artificiallysynthesized linker sequence, 24adp.

SEQ ID NO:4

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HIGLC.

SEQ ID NO:5

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, NHCc2.

SEQ ID NO:6

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, ExcellE.

SEQ ID NO:7

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, ck117.

SEQ ID NO:8

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, M13R.

SEQ ID NO:9

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, 136H.

SEQ ID NO:10

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, 138/9H.

SEQ ID NO:11

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, AILIMHC1.

SEQ ID NO:12

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc1.

SEQ ID NO: 13

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc7.

SEQ ID NO:14

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc8.

SEQ ID NO:15

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc3.

SEQ ID NO:16

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc4.

SEQ ID NO:17

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc6.

SEQ ID NO:18

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HIGHC.

SEQ ID NO:19

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc9.

SEQ ID NO:20

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HCc5.

SEQ ID NO:21

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, polyA.

SEQ ID NO:22

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, AILIMLC1.

SEQ ID NO:23

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, AILIMLC2.

SEQ ID NO:24

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, LCc1.

SEQ ID NO:25

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, LCc2.

SEQ ID NO:26

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence, HIK.

SEQ ID NO:39

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence

SEQ ID NO:40

Other Information: Description of Artificial Sequence: Artificiallysynthesized primer sequence

1. A human antibody which binds to AILIM.
 2. The human antibody of claim1, wherein said AILIM is derived from human.
 3. A human monoclonalantibody which binds to AILIM or a portion thereof.
 4. The humanmonoclonal antibody or a portion thereof of claim 3, wherein said AILIMis derived from human.
 5. The human monoclonal antibody or a portionthereof of claim 3, wherein said human monoclonal antibody has anactivity to inhibit a signal transduction into a cell mediated by AILIM.6. The human monoclonal antibody or a portion thereof of claim 5,wherein said activity to inhibit a signal transduction is (a) or (b) ofthe followings: (a) activity to inhibit proliferation ofAILIM-expressing cells, or (b) activity to inhibit cytokine productionfrom AILIM-expressing cells.
 7. The human monoclonal antibody or aportion thereof of claim 6, wherein said cytokine is one of thecytokines produced by Th1-type or Th2-type T cell.
 8. The humanmonoclonal antibody or a portion thereof of claim 7, wherein saidcytokine is interferon γ or interleukin
 4. 9. The human monoclonalantibody or a portion thereof of claim 5, wherein said human monoclonalantibody has an activity to prevent mixed lymphocyte reaction.
 10. Thehuman monoclonal antibody or a portion thereof of claim 3, wherein saidhuman monoclonal antibody has an activity to induce signal transductioninto a cell mediated by AILIM.
 11. The human monoclonal antibody or aportion thereof of claim 10, wherein said activity to induce signaltransduction is (a) or (b) of the followings: (a) activity to induceproliferation of AILIM-expressing cells, or (b) activity to inducecytokine production from AILIM-expressing cells.
 12. The humanmonoclonal antibody or a portion thereof of claim 11, wherein saidcytokine is one of the cytokines produced by Th1-type or Th2-type Tcell.
 13. The human monoclonal antibody or a portion thereof of claim12, wherein said cytokine is interferon γ or interleukin
 4. 14. Thehuman monoclonal antibody or a portion thereof of claim 3, wherein saidhuman monoclonal antibody has an activity to induce antibody-dependentcytotoxicity to AILIM-expressing cells, and/or immune cytolysis orapoptosis of AILIM-expressing cells.
 15. The human monoclonal antibodyor a portion thereof of claim 3, wherein the binding rate constant (ka)between said monoclonal antibody and AILIM is 1.0×10³ (1/M.Sec) or more.16. The human monoclonal antibody or a portion thereof of claim 15,wherein said binding rate constant (ka) is 1.0×10⁴ (1/M.Sec) or more.17. The human monoclonal antibody or a portion thereof of claim 16,wherein said binding rate constant (ka) is 1.0×10⁵ (1/M.Sec) or more.18. The human monoclonal antibody or a portion thereof of claim 3,wherein the dissociation rate constant (kd) between said monoclonalantibody and AILIM is 1.0×10⁻³ (1/Sec) or less.
 19. The human monoclonalantibody or a portion thereof of claim 18, wherein said dissociationrate constant (kd) is 1.0×10⁻⁴ (1/Sec) or less.
 20. The human monoclonalantibody or a portion thereof of claim 19, wherein said dissociationrate constant (kd) is 1.0×10⁻⁵ (1/Sec) or less.
 21. The human monoclonalantibody or a portion thereof of claim 3, wherein the dissociationconstant (Kd) between said monoclonal antibody and AILIM is 1.0×10⁻⁶ (M)or less.
 22. The human monoclonal antibody or a portion thereof of claim21, wherein said dissociation constant (Kd) is 1.0×10⁻⁷ (M) or less. 23.The human monoclonal antibody or a portion thereof of claim 22, whereinsaid dissociation constant (Kd) is 1.0×10⁻⁸ (M) or less.
 24. The humanmonoclonal antibody or a portion thereof of claim 23, wherein saiddissociation constant (Kd) is 1.0×10⁻⁹ (M) or less.
 25. The humanmonoclonal antibody or a portion thereof of claim 4, wherein a V regionDNA encoding a heavy chain variable region of said human monoclonalantibody is derived from either the human immunoglobulin heavy chain Vgene segment 1-02 or 3-13.
 26. The human monoclonal antibody or aportion thereof of claim 4, wherein a V region DNA encoding a lightchain variable region of said human monoclonal antibody is derived fromeither the human immunoglobulin light chain V gene segment L5 or A27.27. The human monoclonal antibody or a portion thereof of claim 25,wherein a V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from either the humanimmunoglobulin heavy chain V gene segment 1-02 or 3-13, and wherein a Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinlight chain V gene segment L5 or A27.
 28. The human monoclonal antibodyor a portion thereof of claim 27, wherein the V region DNA encoding aheavy chain variable region of said human monoclonal antibody is derivedfrom the human immunoglobulin heavy chain V gene segment 1-02, and the Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from the human immunoglobulin light chainV gene segment L5.
 29. The human monoclonal antibody or a portionthereof of claim 27, wherein the V region DNA encoding a heavy chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin heavy chain V gene segment 3-13, and the V regionDNA encoding a light chain variable region of said human monoclonalantibody is derived from the human immunoglobulin light chain V genesegment A27.
 30. The human monoclonal antibody or a portion thereof ofclaim 4, wherein a heavy chain variable region of said human monoclonalantibody has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 20 through 117 of SEQ ID NO:28, (b) amino acid sequencecomprising amino acids from position 20 through 117 of SEQ ID NO:28 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 20 through 116 of SEQID NO:32, (d) amino acid sequence comprising amino acids from position20 through 116 of SEQ ID NO:32 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 20 through 116 of SEQ ID NO:36, or (f) amino acid sequencecomprising amino acids from position 20 through 116 of SEQ ID NO:36, inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 31. Thehuman monoclonal antibody or a portion thereof of claim 4, wherein aheavy chain polypeptide of said human monoclonal antibody has an aminoacid sequence defined in any of the following (a) through (f): (a) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:28, (b) amino acid sequence comprising amino acids from position20 through 470 of SEQ ID NO:28 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (c) amino acid sequence comprising amino acidsfrom position 20 through 470 of SEQ ID NO:32, (d) amino acid sequencecomprising amino acids from position 20 through 470 of SEQ ID NO:32 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (e) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:36, or (f) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:36 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added.
 32. The human monoclonal antibody or aportion thereof of claim 4, wherein a light chain variable region ofsaid human monoclonal antibody has an amino acid sequence defined in anyof the following (a) through (f): (a) amino acid sequence comprisingamino acids from position 23 through 116 of SEQ ID NO:30, (b) amino acidsequence comprising amino acids from position 23 through 116 of SEQ IDNO:30 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (c) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:34, (d) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:34 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added. (e) amino acid sequencecomprising amino acids from position 21 through 116 of SEQ ID NO:38, or(f) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:38 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.
 33. The human monoclonal antibody or a portion thereof ofclaim 4, wherein a light chain polypeptide of said human monoclonalantibody has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 23 through 236 of SEQ ID NO:30, (b) amino acid sequencecomprising amino acids from position 23 through 236 of SEQ ID NO:30 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 21 through 236 of SEQID NO:34, (d) amino acid sequence comprising amino acids from position21 through 236 of SEQ ID NO:34 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 21 through 236 of SEQ ID NO:38, or (f) amino acid sequencecomprising amino acids from position 21 through 236 of SEQ ID NO:38 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 34. Thehuman monoclonal antibody or a portion thereof of claim 4, wherein saidhuman monoclonal antibody has the following characteristics (a) and (b):(a) a heavy chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 20 through 117 according to SEQID NO:28, and (b) a light chain variable region has an amino acidsequence comprising the amino acid sequence from amino acid 23 through116 according to SEQ ID NO:30.
 35. The human monoclonal antibody or aportion thereof of claim 4, wherein said human monoclonal antibody hasthe following characteristics (a) and (b): (a) a heavy chain polypeptidehas an amino acid sequence from amino acid 20 through 470 according toSEQ ID NO:28, and (b) a light chain polypeptide has an amino acidsequence from amino acid 23 through 236 according to SEQ ID NO:30. 36.The human monoclonal antibody or a portion thereof of claim 4, whereinsaid human monoclonal antibody has the following characteristics (a) and(b): (a) a heavy chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 116according to SEQ ID NO:32, and (b) a light chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid21 through 116 according to SEQ ID NO:34.
 37. The human monoclonalantibody or a portion thereof of claim 4, wherein said human monoclonalantibody has the following characteristics (a) and (b): (a) a heavychain polypeptide has an amino acid sequence comprising the amino acidsequence from amino acid 20 through 470 according to SEQ ID NO:32, and(b) a light chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 21 through 236 according to SEQ IDNO:34.
 38. The human monoclonal antibody or a portion thereof of claim4, wherein said human monoclonal antibody has the followingcharacteristics (a) and (b): (a) a heavy chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid20 through 116 according to SEQ ID NO:36, and (b) a light chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 21 through 116 according to SEQ ID NO:38.
 39. The humanmonoclonal antibody or a portion thereof of claim 4, wherein said humanmonoclonal antibody has the following characteristics (a) and (b): (a) aheavy chain polypeptide has an amino acid sequence comprising the aminoacid sequence from amino acid 20 through 470 according to SEQ ID NO:36,and (b) a light chain polypeptide has an amino acid sequence comprisingthe amino acid sequence from amino acid 21 through 236 according to SEQID NO:38.
 40. The human monoclonal antibody or a portion thereof ofclaim 3, wherein said human monoclonal antibody is a monoclonal antibodyderived from a transgenic non-human mammal capable of producing humanantibodies.
 41. The human monoclonal antibody or a portion thereof ofclaim 40, wherein said human monoclonal antibody is obtained byimmunizing transgenic non-human mammal capable of producing humanantibody with AILIM-expressing cells, membrane fractions derived fromsaid cells, whole molecules constituting AILIM or a portion thereof, orgenes encoding AILIM or a portion thereof.
 42. The human monoclonalantibody or a portion thereof of claim 40, wherein said transgenicnon-human mammal is a transgenic mouse.
 43. A DNA or a portion thereofencoding a polypeptide selected from the group consisting of (a) through(f) below: (a) a polypeptide comprising the amino acid sequence fromamino acid 20 through 117 according to SEQ ID NO:28, (b) a polypeptidecomprising the amino acid sequence from amino acid 23 through 116according to SEQ ID NO:30, (c) a polypeptide comprising the amino acidsequence from amino acid 20 through 116 according to SEQ ID NO:32, (d) apolypeptide comprising the amino acid sequence from amino acid 21through 116 according to SEQ ID NO:34, (e) a polypeptide comprising theamino acid sequence from amino acid 20 through 116 according to SEQ IDNO:36, and (f) a polypeptide comprising the amino acid sequence fromamino acid 21 through 116 according to SEQ ID NO:38.
 44. A DNA or aportion thereof encoding a polypeptide selected from the groupconsisting of (a) through (f) below: (a) a polypeptide comprising theamino acid sequence from amino acids 20 through 470 according to SEQ IDNO:28, (b) a polypeptide comprising the amino acid sequence from aminoacids 23 through 236 according to SEQ ID NO:30, (c) a polypeptidecomprising the amino acid sequence from amino acids 20 through 470according to SEQ ID NO:32, (d) a polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34, (e)a polypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:36, and (f) a polypeptide comprisingthe amino acid sequence from amino acids 21 through 236 according to SEQID NO:38.
 45. A DNA or a portion thereof selected from the groupconsisting of (a) through (f) below: (a) a DNA comprising the nucleotidesequence from nucleotides 126 through 419 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 105 through386 according to SEQ ID NO:29, (c) a DNA comprising the nucleotidesequence from nucleotides 151 through 441 according to SEQ ID NO:31, (d)a DNA comprising the nucleotide sequence from nucleotides 88 through 375according to SEQ ID NO:33, (e) a DNA comprising the nucleotide sequencefrom nucleotides 153 through 443 according to SEQ ID NO:35, and (f) aDNA comprising the nucleotide sequence from nucleotides 93 through 380according to SEQ ID NO:37.
 46. A DNA or a portion thereof selected froma group consisting of (a) through (f) below: (a) a DNA comprising thenucleotide sequence from nucleotides 69 through 1481 according to SEQ IDNO:27, (b) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29, (c) a DNA comprising thenucleotide sequence from nucleotides 94 through 1506 defined in SEQ IDNO:31, (d) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33, (e) a DNA comprising thenucleotide sequence from nucleotides 96 through 1508 according to SEQ IDNO:35, and (f) a DNA comprising the nucleotide sequence from nucleotides33 through 743 according to SEQ ID NO:37.
 47. A vector comprising theDNA of claim
 43. 48. The vector of claim 47 comprising a DNA accordingto any of the following (a) through (c): (a) a DNA comprising thenucleotide sequence from nucleotides 126 through 419 according to SEQ IDNO:27, (b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, or (c) a DNA comprising thenucleotide sequence from nucleotides 153 through 443 according to SEQ IDNO:35.
 49. The vector of claim 47 comprising a DNA according to any ofthe following (a) through (c): (a) a DNA comprising the nucleotidesequence from nucleotides 69 through 1481 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, or (c) a DNA comprising the nucleotidesequence from nucleotides 96 through 1508 according to SEQ ID NO:35. 50.The vector of claim 47 comprising a DNA according to any of thefollowing (a) through (c): (a) a DNA comprising the nucleotide sequencefrom nucleotides 105 through 386 according to SEQ ID NO:29, (b) a DNAcomprising the nucleotide sequence from nucleotides 88 through 375according to SEQ ID NO:33, or (c) a DNA comprising the nucleotidesequence from nucleotides 93 through 380 according to SEQ ID NO:37. 51.The vector of claim 47 comprising a DNA according to any of thefollowing (a) through (c): (a) a DNA comprising the nucleotide sequencefrom nucleotides 39 through 749 according to SEQ ID NO:29, (b) a DNAcomprising the nucleotide sequence from nucleotides 28 through 738according to SEQ ID NO:33, or (c) a DNA comprising the nucleotidesequence from nucleotides 33 through 743 according to SEQ ID NO:37. 52.The vector of claim 47 comprising a DNA according to the following (a)and (b): (a) a DNA comprising the nucleotide sequence from nucleotides126 through 419 according to SEQ ID NO:27, and (b) a DNA comprising thenucleotide sequence from nucleotides 105 through 386 according to SEQ IDNO:29.
 53. The vector of claim 47 comprising a DNA according to thefollowing (a) and (b): (a) a DNA comprising the nucleotide sequence fromnucleotides 69 through 1481 according to SEQ ID NO:27, and (b) a DNAcomprising the nucleotide sequence from nucleotides 39 through 749according to SEQ ID NO:29.
 54. The vector of claim 47 comprising a DNAaccording to the following (a) and (b): (a) a DNA comprising thenucleotide sequence from nucleotides 151 through 441 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides88 through 375 according to SEQ ID NO:33.
 55. The vector of claim 47comprising a DNA according to the following (a) and (b): (a) a DNAcomprising the nucleotide sequence from nucleotides 94 through 1506according to SEQ ID NO:31, and (b) a DNA comprising the nucleotidesequence from nucleotides 28 through 738 according to SEQ ID NO:33. 56.The vector of claim 47 comprising a DNA according to the following (a)and (b): (a) a DNA comprising the nucleotide sequence from nucleotides153 through 443 according to SEQ ID NO:35, and (b) a DNA comprising thenucleotide sequence from nucleotides 93 through 380 according to SEQ IDNO:37.
 57. The vector of claim 47 comprising a DNA according to thefollowing (a) and (b): (a) a DNA comprising the nucleotide sequence fromnucleotides 96 through 1508 according to SEQ ID NO:35, and (b) a DNAcomprising the nucleotide sequence from nucleotides 33 through 743according to SEQ ID NO:37.
 58. A cell producing a human monoclonalantibody of claim
 3. 59. The cell of claim 58, wherein said cell is afused cell obtained by fusing B cell, derived from a mammal capable ofproducing said human monoclonal antibody, and myeloma cell derived froma mammal.
 60. A genetic recombinant host transformed by transferring aDNA described below in (a) or a vector comprising said DNA, a DNAdescribed below in (b) or a vector comprising said DNA, or both DNAsdescribed below in (a) and (b) or a vector comprising both of said DNAs:(a) a DNA encoding a heavy chain polypeptide or a portion thereof of amonoclonal antibody which binds to human AILIM; or (b) a DNA encoding alight chain polypeptide or a portion thereof of a monoclonal antibodywhich binds to human AILIM.
 61. The genetic recombinant host of claim60, wherein said monoclonal antibody is a human monoclonal antibody. 62.The genetic recombinant host of claim 60, wherein said host is amammalian cell.
 63. The genetic recombinant host of claim 60, whereinsaid host is a mammalian fertilized egg.
 64. The genetic recombinanthost of claim 60, wherein said heavy chain polypeptide is one of theheavy chain polypeptides selected from the group consisting of thefollowing (a) through (c): (a) a heavy chain polypeptide comprising theamino acid sequence from amino acids 20 through 117 according to SEQ IDNO:28, (b) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO:32, and (c) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 116 according to SEQ ID NO:36.
 65. The geneticrecombinant host of claim 60, wherein said heavy chain polypeptide isone of the heavy chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a heavy chain polypeptide comprisingthe amino acid sequence from amino acids 20 through 470 according to SEQID NO:28, (b) a heavy chain polypeptide comprising the amino acidsequence from amino acids 20 through 470 according to SEQ ID NO:32, and(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:36.
 66. The geneticrecombinant host of claim 60, wherein said light chain polypeptide isone of the light chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a heavy chain polypeptide comprisingthe amino acid sequence from amino acids 23 through 116 according to SEQID NO:30, (b) a heavy chain polypeptide comprising the amino acidsequence from amino acids 21 through 116 according to SEQ ID NO:34, and(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.
 67. The geneticrecombinant host of claim 60, wherein said light chain polypeptide isone of the light chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a light chain polypeptide comprisingthe amino acid sequence from amino acids 23 through 236 according to SEQID NO:30, (b) a light chain polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34, and(c) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:38.
 68. The geneticrecombinant host of claim 60, wherein said heavy chain and light chainpolypeptides are those defined below in (a) and (b), respectively: (a) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 117 according to SEQ ID NO:28, and (b) a light chainpolypeptide comprising the amino acid sequence from amino acids 23through 116 according to SEQ ID NO:30.
 69. The genetic recombinant hostof claim 60, wherein said heavy chain and light chain polypeptides arethose defined below in (a) and (b), respectively: (a) a heavy chainpolypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:28, and (b) a light chain polypeptidecomprising the amino acid sequence from amino acids 23 through 236according to SEQ ID NO:30.
 70. The genetic recombinant host of claim 60,wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively: (a) a heavy chain polypeptidecomprising the amino acid sequence from amino acids 20 through 116according to SEQ ID NO:32, and (b) a light chain polypeptide comprisingthe amino acid sequence from amino acids 21 through 116 according to SEQID NO:34.
 71. The genetic recombinant host of claim 60, wherein saidheavy chain and light chain polypeptides are those defined below in (a)and (b), respectively: (a) a heavy chain polypeptide comprising theamino acid sequence from amino acids 20 through 470 according to SEQ IDNO:32, and (b) a light chain polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34. 72.The genetic recombinant host of claims 60, wherein said heavy chain andlight chain polypeptides are those defined below in (a) and (b),respectively: (a) a heavy chain polypeptide comprising the amino acidsequence from amino acids 20 through 116 according to SEQ ID NO:36, and(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.
 73. The geneticrecombinant host of claim 60, wherein said heavy chain and light chainpolypeptides are those defined below in (a) and (b), respectively: (a) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 470 according to SEQ ID NO:36, and (b) a light chainpolypeptide comprising the amino acid sequence from amino acids 21through 236 according to SEQ ID NO:38.
 74. The genetic recombinant hostof claim 60, wherein the DNA encoding said heavy chain polypeptide is aDNA defined in any of following (a) through (c): (a) a DNA comprisingthe nucleotide sequence from nucleotides 126 through 419 according toSEQ ID NO:27, (b) a DNA comprising the nucleotide sequence fromnucleotides 151 through 441 according to SEQ ID NO:31, and (c) a DNAcomprising the nucleotide sequence from nucleotides 153 through 443according to SEQ ID NO:35.
 75. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is a DNA definedin any of following (a) through (c): (a) a DNA comprising the nucleotidesequence from nucleotides 69 through 1481 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, and (c) a DNA comprising the nucleotidesequence from nucleotides 96 through 1508 according to SEQ ID NO:35. 76.The genetic recombinant host of claim 60, wherein the DNA encoding saidlight chain polypeptide is a DNA defined in any of following (a) through(c): (a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29, (b) a DNA comprising thenucleotide sequence from nucleotides 88 through 375 according to SEQ IDNO:33, and (c) a DNA comprising the nucleotide sequence from nucleotides93 through 380 according to SEQ ID NO:37.
 77. The genetic recombinanthost of claim 60, wherein the DNA encoding said light chain polypeptideis a DNA as defined in any of following (a) through (c): (a) a DNAcomprising the nucleotide sequence from nucleotides 39 through 749according to SEQ ID NO:29, (b) a DNA comprising the nucleotide sequencefrom nucleotides 28 through 738 according to SEQ ID NO:33, and (c) a DNAcomprising the nucleotide sequence from nucleotides 33 through 743according to SEQ ID NO:37.
 78. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is a DNA describedbelow in (a), and the DNA encoding said light chain polypeptide is a DNAas described below in (b): (a) a DNA comprising the nucleotide sequencefrom nucleotides 126 through 419 according to SEQ ID NO:27, and (b) aDNA comprising the nucleotide sequence from nucleotides 105 through 386according to SEQ ID NO:29.
 79. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 69 through 1481 according to SEQ IDNO:27, and (b) a DNA comprising the nucleotide sequence from nucleotides39 through 749 according to SEQ ID NO:29.
 80. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 151 through 441 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides88 through 375 V SEQ ID NO:33.
 81. The genetic recombinant host of claim60, wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 94 through 1506 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides28 through 738 according to SEQ ID NO:33.
 82. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 153 through 443 according to SEQ IDNO:35, and (b) a DNA comprising the nucleotide sequence from nucleotides93 through 380 according to SEQ ID NO:37.
 83. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 96 through 1508 according to SEQ IDNO:35, and (b) a DNA comprising the nucleotide sequence from nucleotides33 through 743 according to SEQ ID NO:37.
 84. A human monoclonalantibody or a portion thereof produced by a genetic recombinant host(provided excluding the case where said host is a fertilized egg) ofclaim
 60. 85. A pharmaceutical composition comprising the human antibodyof claim 1, and a pharmaceutically acceptable carrier.
 86. Apharmaceutical composition comprising the human monoclonal antibody or aportion thereof of claim 3, and a pharmaceutically acceptable carrier.87. A pharmaceutical composition comprising a human monoclonal antibodyor a portion thereof of claim 84, and a pharmaceutically acceptablecarrier.
 88. The pharmaceutical composition of claim 85, wherein saidpharmaceutical composition is used to inhibit signal transduction intothe cell mediated by AILIM.
 89. The pharmaceutical composition of claim85, wherein said pharmaceutical composition is used to preventproliferation of AILIM-expressing cells.
 90. The pharmaceuticalcomposition of claim 85, wherein said pharmaceutical composition is usedto prevent production of a cytokine from AILIM-expressing cells.
 91. Thepharmaceutical composition of claim 85, wherein said pharmaceuticalcomposition is used to induce signal transduction into a cell mediatedby AILIM.
 92. The pharmaceutical composition of claim 85, wherein saidpharmaceutical composition is used to induce proliferation ofAILIM-expressing cells.
 93. The pharmaceutical composition of claim 85,wherein said pharmaceutical composition is used to induce production ofa cytokine from AILIM-expressing cells.
 94. The pharmaceuticalcomposition of claim 85, wherein said pharmaceutical composition is usedto induce antibody-dependent cytotoxicity against AILIM-expressingcells, and/or immune cytolysis or apoptosis of AILIM-expressing cells.95. A pharmaceutical composition for preventing, treating, orprophylaxis of delayed type allergy, comprising a substance having anactivity in modulating signal transduction mediated by AILIM, and apharmaceutically acceptable carrier.
 96. The pharmaceutical compositionof claim 95, wherein the substance is a protein substance.
 97. Thepharmaceutical composition of claim 96, wherein the protein substance isselected from the group consisting of: a) an antibody which binds toAILIM or a portion thereof; b) a polypeptide comprising the whole or aportion of an extracellular region of AILIM; c) a fusion polypeptidecomprising the whole or a portion of an extracellular region of AILIM,and the whole or a portion of a constant region of immunoglobulin heavychain; and d) a polypeptide which binds to AILIM.
 98. The pharmaceuticalcomposition of claim 97, wherein said antibody that binds to AILIM is ahuman antibody.
 99. The pharmaceutical composition of claim 97, whereinsaid antibody that binds to AILIM is a human monoclonal antibody. 100.The pharmaceutical composition of claim 97, wherein said antibodyagainst AILIM is a human monoclonal antibody produced by a geneticrecombinant host transformed by transferring a DNA described below in(a) or a vector comprising said DNA, a DNA described below in (b) or avector comprising said DNA, or both DNAs described below in (a) and (b)or a vector comprising both of said DNAs: (a) a DNA encoding a heavychain polypeptide or a portion thereof of a monoclonal antibody whichbinds to human AILIM; or (b) a DNA encoding a light chain polypeptide ora portion thereof of a monoclonal antibody which binds to human AILIM.101. The pharmaceutical composition of claim 95, wherein the substanceis a non-protein substance.
 102. The pharmaceutical composition of claim101, wherein the non-protein substance is DNA, RNA, or a chemicallysynthesized compound.
 103. A method for identifying substances that bindto AILIM or AILIM ligand comprising the following processes: (a)preparing an insoluble carrier on which the entire extracellular regionof AILIM or a portion thereof is immobilized; (b) preparing apolypeptide comprising the whole extracellular region of AILIM ligand ora portion thereof labeled with a labeling material that emit adetectable signal; (c) reacting the insoluble carrier in process(a) withthe polypeptide in process (b); (d) reacting the insoluble carrier ofprocess (a), the polypeptide of process (b) and said substance to eachother in any arbitrary orders; (e) detecting the signal emitted fromsaid labeling material contained in the complex produced in process (c),and the signal emitted from said labeling material contained in thecomplex produced in process (d), respectively; and (f) comparing themagnitude of each of signals detected in process (e).
 104. A method foridentifying substances that bind to AILIM or AILIM ligand comprising thefollowing processes: (a) preparing an insoluble carrier on which theentire extracellular region of AILIM ligand or a portion thereof isimmobilized; (b) preparing a polypeptide comprising the wholeextracellular region of AILIM or a portion thereof labeled with alabeling material that emit a detectable signal; (c) reacting theinsoluble carrier in process (a) with the polypeptide in process (b);(d) reacting the insoluble carrier of process (a), the polypeptide ofprocess (b) and said substance to each other in any arbitrary orders;(e) detecting the signal emitted from said labeling material containedin the complex produced in process (c), and the signal emitted from saidlabeling material contained in the complex produced in process (d),respectively; and (f) comparing the magnitude of each of signalsdetected in process (e).
 105. The method of claim 103, wherein saidpolypeptide comprising the whole extracellular region of AILIM or aportion thereof is a fusion polypeptide comprising a polypeptide,comprising the whole extracellular region of AILIM or a portion thereof,and the whole constant region of immunoglobulin heavy chain or a portionthereof.
 106. The method of claim 103, wherein said polypeptidecomprising the whole extracellular region of AILIM ligand or a portionthereof is a fusion polypeptide comprising a polypeptide, comprising thewhole extracellular region of AILIM ligand or a portion thereof, and thewhole constant region of immunoglobulin heavy chain or a portionthereof.
 107. The method of claim 103, wherein said AILIM is a humanAILIM.
 108. The method of claim 103, wherein said AILIM ligand is ahuman AILIM ligand.
 109. A method of inhibiting AILIM-mediated signaltransduction, preventing proliferation of AILIM-expressing cells,preventing production of a cytokine from AILIM-expressing cells,inducing AILIM-mediated signal transduction, inducing proliferation ofAILIM-expressing cells, inducing production of a cytokine fromAILIM-expressing cells, inducing antibody-dependent cytotoxicity againstAILIM-expressing cells, or inducing immune cytolysis or apoptosis ofAILIM-expressing cells in a subject, the method comprising administeringto the subject an effective amount of a pharmaceutical compositioncomprising (i) a pharmaceutically acceptable carrier and (ii) a humanantibody or portion thereof that binds to human AILIM.
 110. The methodof claim 109, wherein the binding rate constant (ka) between the humanantibody or portion thereof and AILIM is 1.0×10³ (1/M.Sec) or more,1.0×10⁴ (1/M.Sec) or more, or 1.0×10⁵ (1/M.Sec) or more.
 111. The methodof claim 109, wherein the dissociation rate constant (kd) between thehuman antibody or portion thereof and AILIM is 1.0×10⁻³ (1/Sec) or less,1.0×10⁻⁴ (1/Sec) or less, 1.0×10⁻⁵ (1/Sec) or less, 1.0×10⁻⁶ (M) orless, 1.0×10⁻⁷ (M) or less, 1.0×10⁻⁸ (M) or less, or 1.0×10⁻⁹ (M) orless.
 112. The method of claim 109, wherein: a V region DNA encoding aheavy chain variable region of the human antibody or portion thereof isderived from either the human immunoglobulin heavy chain V gene segment1-02 or 3-13; or a V region DNA encoding a light chain variable regionof the human antibody or portion thereof is derived from either thehuman immunoglobulin light chain V gene segment L5 or A27.
 113. Themethod of claim 112, wherein the V region DNA encoding a heavy chainvariable region of the human antibody or portion thereof is derived fromeither the human immunoglobulin heavy chain V gene segment 1-02 or 3-13,and wherein the V region DNA encoding a light chain variable region ofthe human antibody or portion thereof is derived from either the humanimmunoglobulin light chain V gene segment L5 or A27.
 114. The method ofclaim 112, wherein the V region DNA encoding a heavy chain variableregion of the human antibody or portion thereof is derived from thehuman immunoglobulin heavy chain V gene segment 1-02, and the V regionDNA encoding a light chain variable region of the human antibody orportion thereof is derived from the human immunoglobulin light chain Vgene segment L5.
 115. The method of claim 112, wherein the V region DNAencoding a heavy chain variable region of the human antibody or portionthereof is derived from the human immunoglobulin heavy chain V genesegment 3-13, and the V region DNA encoding a light chain variableregion of the human antibody or portion thereof is derived from thehuman immunoglobulin light chain V gene segment A27.
 116. The method ofclaim 109, wherein a heavy chain variable region of the human antibodyor portion thereof has an amino acid sequence defined in any of thefollowing (a) through (f): (a) amino acid sequence comprising aminoacids from position 20 through 117 of SEQ ID NO:28, (b) amino acidsequence comprising amino acids from position 20 through 117 of SEQ IDNO:28 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (c) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:32, (d) amino acid sequence comprising aminoacids from position 20 through 116 of SEQ ID NO:32 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added. (e) amino acid sequencecomprising amino acids from position 20 through 116 of SEQ ID NO:36, or(f) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:36, in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.
 117. The method of claim 109, wherein a heavy chainpolypeptide of the human antibody or portion thereof has an amino acidsequence defined in any of the following (a) through (f): (a) amino acidsequence comprising amino acids from position 20 through 470 of SEQ IDNO:28, (b) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:28 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added. (c) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:32, (d) amino acid sequencecomprising amino acids from position 20 through 470 of SEQ ID NO:32 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (e) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:36, or (f) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:36 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added.
 118. The method of claim 109, wherein alight chain variable region of the human antibody or portion thereof hasan amino acid sequence defined in any of the following (a) through (f):(a) amino acid sequence comprising amino acids from position 23 through116 of SEQ ID NO:30, (b) amino acid sequence comprising amino acids fromposition 23 through 116 of SEQ ID NO:30 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added. (c) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:34, (d) amino acidsequence comprising amino acids from position 21 through 116 of SEQ IDNO:34 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (e) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:38, or (f) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:38 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added.
 119. The method of claim 109,wherein a light chain polypeptide of the human antibody or portionthereof has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 23 through 236 of SEQ ID NO:30, (b) amino acid sequencecomprising amino acids from position 23 through 236 of SEQ ID NO:30 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 21 through 236 of SEQID NO:34, (d) amino acid sequence comprising amino acids from position21 through 236 of SEQ ID NO:34 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 21 through 236 of SEQ ID NO:38, or (f) amino acid sequencecomprising amino acids from position 21 through 236 of SEQ ID NO:38 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 120. Themethod of claim 109, wherein the human antibody or portion thereof hasthe following characteristics (a) and (b): (a) a heavy chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 20 through 117 according to SEQ ID NO:28, and (b) alight chain variable region has an amino acid sequence comprising theamino acid sequence from amino acid 23 through 116 according to SEQ IDNO:30.
 121. The method of claim 109, wherein the human antibody orportion thereof has the following characteristics (a) and (b): (a) aheavy chain polypeptide has an amino acid sequence from amino acid 20through 470 according to SEQ ID NO:28, and (b) a light chain polypeptidehas an amino acid sequence from amino acid 23 through 236 according toSEQ ID NO:30.
 122. The method of claim 109, wherein the human antibodyor portion thereof has the following characteristics (a) and (b): (a) aheavy chain variable region has an amino acid sequence comprising theamino acid sequence from amino acid 20 through 116 according to SEQ IDNO:32, and (b) a light chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 116according to SEQ ID NO:34.
 123. The method of claim 109, wherein thehuman antibody or portion thereof has the following characteristics (a)and (b): (a) a heavy chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 470according to SEQ ID NO:32, and (b) a light chain polypeptide has anamino acid sequence comprising the amino acid sequence from amino acid21 through 236 according to SEQ ID NO:34.
 124. The method of claim 109,wherein the human antibody or portion thereof has the followingcharacteristics (a) and (b): (a) a heavy chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid20 through 116 according to SEQ ID NO:36, and (b) a light chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 21 through 116 according to SEQ ID NO:38.
 125. Themethod of claim 109, wherein the human antibody or portion thereof hasthe following characteristics (a) and (b): (a) a heavy chain polypeptidehas an amino acid sequence comprising the amino acid sequence from aminoacid 20 through 470 according to SEQ ID NO:36, and (b) a light chainpolypeptide has an amino acid sequence comprising the amino acidsequence from amino acid 21 through 236 according to SEQ ID NO:38. 126.A method of treating or preventing rheumatoid arthritis, multiplesclerosis, autoimmune thyroiditis, allergic contact dermatitis, lichenplanus as a chronic inflammatory skin disease, systemic lupuserythematosus, insulin dependent diabetes mellitus, psoriasis,autoimmune diseases, allergic diseases, delayed type allergy,arthropathies, rheumatoid arthritis, osteoarthritis, inflammation,hepatitis, graft versus host reaction, graft versus host disease,immunorejection associated with transplantation of tissues or organs,immune response to foreign antigen or self antigen, diseases caused byabnormality in gut immunity, inflammatory bowel diseases, Crohn'sdisease, ulcerative colitis, alimentary allergy, inflammation associatedwith arthritides, inflammation associated with rheumatoid arthritis,inflammation associated with osteoarthritis, pneumonia, inflammationassociated with infectious diseases, enteritis, nephritis, glomerularnephritis, inflammation associated with kidney fibrosis, gastritis,vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis,encephalitis, inflammation associated with ischemia-reperfusion injury,inflammation associated with myocardial ischemia-reperfusion injury,inflammation associated with immunorejection after transplantation oftissues or organs, scald, skin inflammations, inflammation associatedwith multiple organ failure, inflammation after operation of PTCA orPTCR, inflammation associated with atherosclerosis, or inflammationassociated with autoimmune thyroiditis, the method comprisingadministering to a subject an effective amount of a pharmaceuticalcomposition comprising (i) a pharmaceutically acceptable carrier and(ii) a human antibody or portion thereof that binds to human AILIM. 127.The method of claim 126, wherein the binding rate constant (ka) betweenthe human antibody or portion thereof and AILIM is 1.0×10³ (1/M.Sec) ormore, 1.0×10⁴ (1/M.Sec) or more, or 1.0×10⁵ (1/M.Sec) or more.
 128. Themethod of claim 126, wherein the dissociation rate constant (kd) betweenthe human antibody or portion thereof and AILIM is 1.0×10⁻³ (1/Sec) orless, 1.0×10⁻⁴ (1/Sec) or less, 1.0×10⁻⁵ (1/Sec) or less, 1.0×10⁻⁶ (M)or less, 1.0×10⁻⁷ (M) or less, 1.0×10⁻⁸ (M) or less, or 1.0×10⁻⁹ (M) orless.
 129. The method of claim 126, wherein: a V region DNA encoding aheavy chain variable region of the human antibody or portion thereof isderived from either the human immunoglobulin heavy chain V gene segment1-02 or 3-13; or a V region DNA encoding a light chain variable regionof the human antibody or portion thereof is derived from either thehuman immunoglobulin light chain V gene segment L5 or A27.
 130. Themethod of claim 129, wherein the V region DNA encoding a heavy chainvariable region of the human antibody or portion thereof is derived fromeither the human immunoglobulin heavy chain V gene segment 1-02 or 3-13,and wherein the V region DNA encoding a light chain variable region ofthe human antibody or portion thereof is derived from either the humanimmunoglobulin light chain V gene segment L5 or A27.
 131. The method ofclaim 129, wherein the V region DNA encoding a heavy chain variableregion of the human antibody or portion thereof is derived from thehuman immunoglobulin heavy chain V gene segment 1-02, and the V regionDNA encoding a light chain variable region of the human antibody orportion thereof is derived from the human immunoglobulin light chain Vgene segment L5.
 132. The method of claim 129, wherein the V region DNAencoding a heavy chain variable region of the human antibody or portionthereof is derived from the human immunoglobulin heavy chain V genesegment 3-13, and the V region DNA encoding a light chain variableregion of the human antibody or portion thereof is derived from thehuman immunoglobulin light chain V gene segment A27.
 133. The method ofclaim 126, wherein a heavy chain variable region of the human antibodyor portion thereof has an amino acid sequence defined in any of thefollowing (a) through (f): (a) amino acid sequence comprising aminoacids from position 20 through 117 of SEQ ID NO:28, (b) amino acidsequence comprising amino acids from position 20 through 117 of SEQ IDNO:28 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (c) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:32, (d) amino acid sequence comprising aminoacids from position 20 through 116 of SEQ ID NO:32 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added. (e) amino acid sequencecomprising amino acids from position 20 through 116 of SEQ ID NO:36, or(f) amino acid sequence comprising amino acids from position 20 through116 of SEQ ID NO:36, in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.
 134. The method of claim 126, wherein a heavy chainpolypeptide of the human antibody or portion thereof has an amino acidsequence defined in any of the following (a) through (f): (a) amino acidsequence comprising amino acids from position 20 through 470 of SEQ IDNO:28, (b) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:28 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added. (c) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:32, (d) amino acid sequencecomprising amino acids from position 20 through 470 of SEQ ID NO:32 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (e) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:36, or (f) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:36 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added.
 135. The method of claim 126, wherein alight chain variable region of the human antibody or portion thereof hasan amino acid sequence defined in any of the following (a) through (f):(a) amino acid sequence comprising amino acids from position 23 through116 of SEQ ID NO:30, (b) amino acid sequence comprising amino acids fromposition 23 through 116 of SEQ ID NO:30 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added. (c) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:34, (d) amino acidsequence comprising amino acids from position 21 through 116 of SEQ IDNO:34 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (e) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:38, or (f) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:38 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added.
 136. The method of claim 126,wherein a light chain polypeptide of the human antibody or portionthereof has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 23 through 236 of SEQ ID NO:30, (b) amino acid sequencecomprising amino acids from position 23 through 236 of SEQ ID NO:30 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 21 through 236 of SEQID NO:34, (d) amino acid sequence comprising amino acids from position21 through 236 of SEQ ID NO:34 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 21 through 236 of SEQ ID NO:38, or (f) amino acid sequencecomprising amino acids from position 21 through 236 of SEQ ID NO:38 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 137. Themethod of claim 126, wherein the human antibody or portion thereof hasthe following characteristics (a) and (b): (a) a heavy chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 20 through 117 according to SEQ ID NO:28, and (b) alight chain variable region has an amino acid sequence comprising theamino acid sequence from amino acid 23 through 116 according to SEQ IDNO:30.
 138. The method of claim 126, wherein the human antibody orportion thereof has the following characteristics (a) and (b): (a) aheavy chain polypeptide has an amino acid sequence from amino acid 20through 470 according to SEQ ID NO:28, and (b) a light chain polypeptidehas an amino acid sequence from amino acid 23 through 236 according toSEQ ID NO:30.
 139. The method of claim 126, wherein the human antibodyor portion thereof has the following characteristics (a) and (b): (a) aheavy chain variable region has an amino acid sequence comprising theamino acid sequence from amino acid 20 through 116 according to SEQ IDNO:32, and (b) a light chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 116according to SEQ ID NO:34.
 140. The method of claim 126, wherein thehuman antibody or portion thereof has the following characteristics (a)and (b): (a) a heavy chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 470according to SEQ ID NO:32, and (b) a light chain polypeptide has anamino acid sequence comprising the amino acid sequence from amino acid21 through 236 according to SEQ ID NO:34.
 141. The method of claim 126,wherein the human antibody or portion thereof has the followingcharacteristics (a) and (b): (a) a heavy chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid20 through 116 according to SEQ ID NO:36, and (b) a light chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 21 through 116 according to SEQ ID NO:38.
 142. Themethod of claim 126, wherein the human antibody or portion thereof hasthe following characteristics (a) and (b): (a) a heavy chain polypeptidehas an amino acid sequence comprising the amino acid sequence from aminoacid 20 through 470 according to SEQ ID NO:36, and (b) a light chainpolypeptide has an amino acid sequence comprising the amino acidsequence from amino acid 21 through 236 according to SEQ ID NO:38.